RNA纯化的染色质隔离(CHIRP)

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Summary

CHIRP技术是一种新型快速映射约束力的长非编码RNA基因组网站(lncRNAs)。该方法采用特异性的反义平铺寡核苷酸允许枚举的lncRNA绑定基因组站点的优势。

Cite this Article

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Chu, C., Quinn, J., Chang, H. Y. Chromatin Isolation by RNA Purification (ChIRP). J. Vis. Exp. (61), e3912, doi:10.3791/3912 (2012).

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Abstract

长非编码RNA的染色质状态的关键调节剂量补偿,压印和发育的基因表达1,2,3,4,5,6,7,如重要的生物过程。最近发现的成千上万的在特定的染色质修饰复合物,如多梳镇压复杂2(PRC2),的关联lnc​​RNAs与介导的组蛋白H3赖氨酸27三甲基化(H3K27me3),建议在管理中的特定基因的染色质状态的众多lncRNAs广泛的角色时尚8,9。虽然一些lncRNAs被认为在邻近基因的顺,其他lncRNAs工作的反式调节基因的远亲位于。例如, 果蝇 lncRNAs roX1和roX2绑定的雄性细胞的X染色体上的许多地区,是至关重要的剂量补偿10,11。然而,他们的结合位​​点的确切位置,不知道在高分辨率。同样,人类的lncRNA热风可以影响对H PRC2占用基因的全基因组3,12,13,但特异性是如何实现的undreds目前还不清楚。 LncRNAs也可以作为模块化脚手架,聘请多蛋白质复合物的组装。经典反义RNA支架是TERC的RNA,由于复杂的14端粒酶模板和脚手架;热风也可以作为一个PRC2脚手架和H3K4的去甲基复杂的13。

此前的研究显示映射在染色的RNA占用大量的见解15,16,但只有一次一个单一的基因位点。占用网站的大多数lncRNAs的是不知道,和染色质调控lncRNAs的角色大多已推断的lncRNA扰动的间接影响。正如染色质免疫芯片或深度测序(芯片的芯片或芯片SEQ,分别)大大改善了我们的基因组规模的蛋白质-DNA相互作用的理解,在这里我们说明了recenTLY出版策略映射在高分辨率17长的RNA入住的全基因组。基于这种方法,通过RNA纯化(CHIRP)( 图1),染色质隔离亲和力捕捉目标lncRNA:染色质复杂,由平铺反义寡核苷酸,然后生成基因组结合位点的地图,在几百基地的决议高灵敏度和低背景。 CHIRP是适用于许多lncRNAs因为亲和探针的设计是简单的RNA序列,并要求没有RNA的结构或功能域的知识。

Protocol

1。探针设计

CHIRP设计反义DNA瓦片目标RNA的选择性检索的探针。

  1. 设计反义寡核苷酸探针在网上探头设计师singlemoleculefish.com 18。
  2. 使用这些参数:探头= 1探针/ 100 bp的RNA的长度; 2)目标GC%= 45; 3)寡核苷酸长度= 20; 4)间隔长度= 60-80。如果太长的设计师闯入段的RNA。省略重复或广泛的同源性区域。
  3. 为了反义DNA探针在3总理结束BiotinTEG。
  4. 根据自己的位置沿RNA标记探针。他们分成两个游泳池,使“甚至”池包含所有探头的编号2,4,6,等“奇”池包含探针编号1,3,5,等到100微米的浓度和稀释的探针池储存于-20°C
  5. 所有的实验都进行两个游泳池,对方的内部控制。真正的RNA依赖的信号会从目前两个池,而探头的噪音会是唯一的每个池。这适用于CHIRP-qPCR和啁啾SEQ。

2。收获细胞

收集细胞CHIRP实验将使用。

  1. 生长在细胞组织培养板或汇合瓶。冲洗用磷酸盐缓冲液(PBS)和t​​rypsinize。淬火胰蛋白酶与媒体的2倍量,吸起来,撞出细胞和悬浮成单细胞悬液。所有媒体和再悬浮细胞转移到50毫升猎鹰管。 20亿个细胞通常足够一CHIRP样品。
  2. 在800RCF 4分钟旋转细胞。抽吸媒体和40毫升的PBS重悬40万个细胞,如果有必要结合管。在800RCF 4分钟旋转细胞。倒出PBS,小心吸的角度上剩余的液体。
  3. 3。交联细胞,收集细胞沉淀

    交联戊二醛保存RNA的染色质相互作用,并准备细胞沉淀收集细胞。

    1. 在室温下执行的所有步骤。
    2. 1%的戊二醛在室温PBS准备。准备10毫升,每10万细胞(0.4毫升25%戊二醛股票+ 9.6毫升PBS)。戊二醛必须使用新鲜。
    3. 点选底部猎鹰管撞出颗粒。重悬细胞沉淀在1%的戊二醛,具有体积小,以避免块开始,然后补足全量。反相混合。在室温下10分钟架桥月底到年底摇床或旋转。
    4. 快速为5分钟,在室温下甘氨酸1.25米的十分之一体积的交联反应。
    5. 在2000RCF离心5分钟。冷藏20毫升PBS一次,在2000RCF纺纱5分钟,吸上清和洗颗粒。
    6. 吸和悬浮在W灰化,交联颗粒与1毫升冷PBS每20万个细胞。每毫升转移到Eppendorf管和自旋为3分钟,4℃枪头删除尽可能多的PBS尽可能仔细2000RCF。
    7. 闪光冻结在-80在液氮和存储单元颗粒°C间下去。

    4。细胞裂解

    裂解交联细胞准备细胞裂解。

    1. 在室温下解冻冷冻细胞颗粒。点击驱逐和混合细胞沉淀。自旋向下沉淀在2000RCF 3分,在4°C用锋利的10μL枪头,以消除任何剩余的PBS。
    2. 在电子天平(精确到1毫克)皮的空Eppendorf管的质量(我们管重1.060克,非常一致)。权衡每个颗粒,并记录其重量。交联的HeLa细胞整整15厘米的菜通常重100毫克。
    3. 补编裂解液(10X球团矿质量,如1毫升100毫克)与FRESĤ蛋白酶抑制剂PMSF和Superase中(见附件缓冲区列表)。拌匀。
    4. 10X量补充裂解液加入每管和悬浮颗粒。对于小颗粒<25毫克,250μL补充裂解液中悬浮。暂停应光滑。如果没有,分为500μL等分的悬挂和使用机动颗粒搅拌机打破团块。立即着手超声。

    5。超声

    通过超声分散交联细胞裂解物的剪切DNA的。

    1. 超声在15毫升猎鹰管细胞裂解Bioruptor。使用<1.5毫升裂解,每管和更快的超声,超声时间不超过两管。
    2. 超声在4℃最高设置30秒的水洗澡ON,OFF脉冲间隔4​​5秒。检查裂解液每30分钟。继续超声分散,不再浑浊,直到细胞裂解。这可能要花费少则30分钟,多达4个小时。数量管,样本量,水浴温度,超声时间内将影响这个过程需要多久。超声管可能会在不同的利率,使他们凝聚在一起,每30分钟重新分配到原管,确保同质化。注:戊二醛交联细胞采取显着更长的时间比甲醛等值超声。
    3. 当裂解变为清晰,5μL的裂解转移到一个新的Eppendorf管中。加入90μL的DNA蛋白酶K(PK),缓冲区(缓冲区列表)和5μL的PK。旋涡混合后短暂离心。孵育45分钟,在50°C。
    4. Qiagen公司的PCR纯化试剂盒提取的DNA。洗脱在30μL的Qiagen公司的洗脱缓冲液(EB)的DNA和1%琼脂糖凝胶电泳检查DNA的大小。如果大量的DNA涂抹100-500基点,超声是完整的。如果没有,继续超声。
    5. 离心10分钟的超声波处理样品在16100RCF 4°C。结合上清,分装成1毫升样品和闪光冻结液体nitrogeN。储存在-80°C。

    6。 CHIRP

    杂交生物素标记的DNA探针,RNA和隔离结合的染色质。

    1. 在室温解冻的染色质管。
    2. 准备杂交缓冲区(缓冲区列表,准备染色毫升,每2毫升)。涡旋混合。
    3. 对于一个典型的的CHIRP使用裂解液1毫升的样品,取出10μLRNA输入和10μL的Eppendorf管中的DNA INPUT和地方。保持在冰上,直到进一步利用。
    4. 1毫升染色质转移至15毫升的猎鹰管。杂交缓冲液中加入2毫升每管。总体积为<1.5毫升,使用Eppendorf管。
    5. 在室温下解冻探头。检查数量,如果你不使用它在很长一段时间(100微米探针的NanoDrop探头规格〜500-600 ng /μl的单链DNA的设置)。具体管(100 pmol探针染色每1毫升,1μL100 pmol /μL的每1毫升染色探头)探头适当的音量。拌匀。在37℃孵育4小时用颤抖。
    6. 随着杂交余下的20分钟,准备的C-1磁珠(储存在4°C)。使用100μL的探头,每100 pmol。 1毫升unsupplemented裂解缓冲液洗三次,使用从缓冲区的DynaMag-2磁铁带单独的珠子。
    7. 原体积的裂解液重悬珠;与新鲜PMSF,有价证券和Superase在补充。经过4小时的杂交反应完成后,每管加100μL珠。拌匀。在37°C孵育30分钟用颤抖。
    8. 准备洗净的缓冲区,每个样品(5毫升)。涡旋混合。预温至37°C。使用前加入PMSF。
    9. 用1毫升洗涤缓冲五倍珠。在第一次洗,使用-15 DynaMag磁条单独的珠子,倒出,并重新悬浮在洗涤缓冲液1毫升。传输量1.5 mL的Eppendorf管中。在37°C孵育5分钟摇晃。
    10. 在随后的洗涤,离心管上每一个minicentrifuge,设置的DynaMag-2 1分钟的磁条上的样本。倒出样品,在洗涤缓冲液1毫升悬浮1 Kimwipe的,任何水滴擦拭。在37°C孵育5分钟摇晃。重复5个总清洗。
    11. 在最后一次洗涤,悬浮珠。取出100μL和RNA分离作废。储备900μLDNA的分数。放在DynaMag-2磁条所有的管子和删除洗缓冲区。后短暂离心管放在磁铁带。用锋利的10μL枪头完全删除最后的洗涤缓冲位。

    7。 RNA分离

    QRT-PCR定量从CHIRP样本中提取的RNA部分。

    1. 以100μL,珠样品和10μLRNA输入采样。加入85μL的RNA PK缓冲液pH 7.0的RNA输入。在95μL,RNA的PK缓冲液pH 7.0重悬珠。在50°C加入5μL,Proteine​​ase K和孵育45分钟月底到年底晃动。
    2. 简单地降速所有的管子,煮沸10分钟的热块样品在95°C。
    3. 冷藏在冰样品,加入500μL的TRIzol试剂,涡旋大力,持续10秒。在室温下孵育10分钟。贮存于-80°C或继续执行步骤4。
    4. 加入100μL氯仿TRIZOL处理的样品。涡大力,持续10秒。在4°C为15分钟的台式离心机上旋转在16100RCF
    5. 删除〜400μL水上清液,避免有机和接口。
    6. 加入600μL(1.5卷)100%的乙醇,并拌匀。旋转样品通过MIRNeasy迷你列。与RWT的(MIRNeasy迷你包),每制造商的协议的视网膜色素上皮2X 1X洗。与30μL无核酸酶H 2 O(NFH 2)洗脱。
    7. 对待每制造商的协议 DNA的RNA洗脱液。反应完成后,样本加热15分钟,在65°C至完全灭活任何剩余的DNA酶。
    8. 使用1μL,RNA的分离定量RT-PCR分析,每口井,以确认lncRNA检索。 GAPDH的经常被用来作为阴性对照。

    8。 DNA提取

    从啁啾样品中提取DNA的部分,经测序,以确定或由定量PCR定量。

    1. 准备DNA洗脱缓冲液(见缓冲区列表),每个样品150μL,包括DNA输入。
    2. 新增10μLRNA酶A(10毫克/毫升)和每毫升DNA洗脱缓冲液10μL,核糖核酸酶H(10 U /μL),涡旋混合。
    3. 悬浮每个样品150μL核糖核酸酶与DNA洗脱缓冲液珠。 (重悬在140μLDNA输入)在37℃孵育30分钟用颤抖。
    4. 单独的珠子和上清DynaMag-2磁条。除去上清液,加入标记的试管。
    5. 准备第二等份的DNA洗脱缓冲液10μL的RNA酶A(10毫克/毫升)和,RNaseH(10的U /μL)8.2完全一样做)。加入150μL,每个样品(包括DNA输入),孵育,弃上清。收集所有的上清液(建议立即进行删除D是〜300μL)。
    6. 加入15μLPK每个样品。在50°C孵育45分钟用颤抖。
    7. 前降速黄色锁相凝胶管(5PRIME)的。锁相凝胶管转移的DNA样本,并添加300μLPhOH:每个样品异戊:氯仿。大力摇晃10分钟,并在16100RCF 5分钟的台式离心机上旋转,在4°C采取水从顶部(约300μL)。新增3 GlycoBlue微升,30μL的醋酸钠,和900μL100%的乙醇。混合和储存在-20°C过夜。
    8. 旋转16100RCF样品在4°C. 30分钟
    9. 小心倒出上清液。加入1毫升70%乙醇,涡旋混合。在16100RCF离心5分钟。取出上清液,用吸管。空气干燥1分钟。悬浮在30μL的光大。
    10. DNA样本分析每Illumina的协议的高通量测序库的qPCR或准备就绪。

    10。代表结果

    图1 图2显示了人类端粒酶RNA(TERC)从HeLa细胞中GAPDH的,丰富的,可作为阴性对照细胞RNA的富集。执行CHIRP拉低TERC的RNA(〜88%)在细胞中的大部分,而只有0.46%的GAPDH RNA检索,展示〜200倍的富集因子。针对LacZ基因的RNA,这是不是在哺乳动物细胞中表达( 图2),探针,如非特异性探针,可以用来作为额外的阴性对照。

    通常富集负地区的qPCR测量时,预计到绑定目标lncRNA的DNA区域。 图3显示四个热风的方向在小学人包皮成纤维细胞的网站,我们通过在同一细胞系的CHIRP SEQ中确定的定量PCR验证,而TERC和GAPDH的DNA位点本身RVE作为阴性对照地区。两个“甚至”和“奇”探头设置产生了负面的地区,真正的lncRNA结合位点的一个标志,预计热风绑定网站可比富集。

    啁啾丰富的DNA高通量测序产生的全球地图lncRNA结合位点。 果蝇的lncRNA roX2是与X染色体交互方式在剂量补偿的要求。 图4显示了一个X染色体的部分roX2约束力的文件。两个“甚至”和“奇”的样品已测序和其独特的声音已被淘汰,产生重叠信号的轨道。每一个“高峰期”,在这里表示强烈的roX2约束力的网站。完整的跟踪和列表roX2靶基因已在楚等人2011 17。

    图1。
    图1。的CHIRP多项式的流程图dure。染色质交联lncRNA: 在体内生物素蛋白加合物平铺探测是杂交lncRNA目标,和染色质复合物使用磁链亲和素磁珠纯化,通过严格的清洗我们洗脱lncRNA结合的DNA或蛋白质的核糖核酸酶A和H鸡尾酒lncRNA一个假定的结合序列,在橙系统化。 2011年以前在楚出版。17

    图2。
    图2。CHIRP丰富,为人类TERC的RNA。 TERC asDNA探头撷取〜蜂窝TERC RNA不到GAPDH的88%。 LacZ基因的asDNA探针作为阴性对照和检索既不的RNA。平均值±SD所示。 2011年以前在楚出版。17

    图3。
    图3。热风CHIRP-qPCR的首要人权埃斯金的成纤维细胞。NFKBIA,HOXD3-4,SERINC5和ABCA2的与热风进行交互的地区。TERCGAPDH服务作为阴性对照。平均值±SD所示。 2011年以前在楚出版。17

    图4。
    图4。CHIRP roX2的RNA-seq的数据在SL2 果蝇细胞。 “即使”和“奇”分别进行测序,他们的数据合并,以反映都只有普通的山峰。合并后的轨道。 2011年以前在楚出版。17

Discussion

在这里,我们介绍了CHIRP-seq技术, 在体内 lncRNA的全基因组结合位点的映射方法。成功的关键参数是平铺的寡核苷酸探针和戊二醛交联的分裂池。亲和探针的设计是简单的RNA序列,无需事先的RNA的结构或功能域的知识。三个相当不同的两个物种的RNA - - 我们与roX2,TERC和热风的成功表明,啁啾seq是可能推广到许多lncRNAs。与所有的实验,护理和适当的控制是需要解释的结果。不同的的lncRNA可能需要滴定的条件,和明智变化的条件,如选择不同的亲和探针或交联剂,可突出RNA的染色质相互作用的不同方面。芯片-seq的一样,并不是所有的绑定事件是必然的功能,需要更多的研究来确定ØRNA的生物后果ccupancy的染色质。尽管如此,我们预计其他的染色相关lncRNAs的,目前在数以万计的数字8,9的研究人员的这项技术的许多有趣的应用程序。正如芯片SEQ打开门的全基因组DNA-蛋白质相互作用的探索,“RNA相互作用组”的CHIRP-SEQ研究的可能揭示生物的许多新的途径。

Disclosures

C.楚和HY张被命名为基于这种方法的专利申请的发明人。

Acknowledgments

我们感谢T.红,三菱商事。蔡O.庄园,E.西格尔,M.黑田东彦,T. Swigut,一讨论Shestopalov。新加坡(CC),国立卫生研究院以R01 CA118750和以R01-HG004361(HYC),加州再生医学研究所(HYC)科学,技术和研究机构的支持。 HYC是霍华德·休斯医学研究所的早期职业科学家。

Materials

Name Company Catalog Number Comments
Buffer List:
Dissolve a pellet of complete protease inhibitor in 1 ml water as 50x stock. Make 100 mM PMSF in isopropanol (100x stock). Superase-in is used as 200x stock. Store all at -20 °C.
Lysis Buffer:
50 mM Tris-Cl pH 7.0
10 mM EDTA
1% SDS
Always add PMSF, P.I. and Superase-in fresh before use except when washing beads
Proteinase K Buffer (for DNA)
100 mM NaCl
10 mM TrisCl pH 8.0 (For RNA use pH 7.0)
1 mM EDTA
0.5% SDS
Add 5% by volume Proteainse K (Ambion AM2546 20 mg/ml) fresh before use
Hybridization Buffer
750 mM NaCl
1% SDS
50 mM Tris-Cl pH 7.0
1 mM EDTA
15% formamide (store in the dark at 4 °C)
Always add PMSF, P.I. and Superase-in fresh before use
Wash Buffer
2x NaCl and Sodium citrate (SSC) (diluted from 20x SSC Invitrogen stock)
0.5% SDS
Always add PMSF fresh before use
DNA elution Buffer
50 mM NaHCO3
1% SDS
Table of specific reagents and equipment:
Glutaraldehyde (EM grade) Sigma-Aldrich G5882-10x10ml
Motorized pellet mixer VWR international V8185-904
Protease inhibitor Roche Group 11873580001
PMSF Sigma-Aldrich 78830
Superase-in Ambion AM2696
Bioruptor Diagenode UCD-200
Falcon tubes (for sonication) Corning 430790
Proteinase K Ambion AM2546
PCR purification kit Qiagen 28106
C-1 magnetic beads Invitrogen 65002
PMSF Sigma-Aldrich P7626-25G
DynaMag-15 magnet Invitrogen 123-01D
DynaMag-2 magnet Invitrogen 123-21D
MIRNeasy mini kit Qiagen 217004
Rnase H Epicentre Biotechnologies R0601K
Rnase A Sigma-Aldrich R4875-100MG
Phase Lock Gel Heavy 5 PRIME 2302810
Trizol Invitrogen 15596-018
Phenol:chloroform:Isoamyl Invitrogen 15593-031
Chloroform Ricca RSOC0020-1C
GlycoBlue Ambion AM9515
Glycine JT Baker 4057-06
PBS, pH 7.4 Invitrogen 10010-049
Elution Buffer (EB) Qiagen 19086
20x SSC Invitrogen 15557-036
10% SDS Invitrogen 15553-027
DNA-free Ambion AM1906
Buffer kit Ambion AM9010
Formamide Invitrogen 15515-026

DOWNLOAD MATERIALS LIST

References

  1. Koziol, M. J., Rinn, J. L. RNA traffic control of chromatin complexes. Curr. Opin. Genet. Dev. 20, 142-148 (2010).
  2. Mercer, T. R., Dinger, M. E., Mattick, J. S. Long non-coding RNAs: insights into functions. Nat. Rev. Genet. 10, 155-159 (2009).
  3. Rinn, J. L. Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell. 129, 1311-1323 (2007).
  4. Zhao, J., Sun, B. K., Erwin, J. A., Song, J. J., Lee, J. T. Polycomb proteins targeted by a short repeat RNA to the mouse X chromosome. Science. 322, 750-756 (2008).
  5. Kelley, R. L. Epigenetic spreading of the Drosophila dosage compensation complex from roX RNA genes into flanking chromatin. Cell. 98, 513-522 (1999).
  6. Pandey, R. R. Kcnq1ot1 antisense noncoding RNA mediates lineage-specific transcriptional silencing through chromatin-level regulation. Mol. Cell. 32, 232-246 (2008).
  7. Wang, K. C. A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression. Nature. 472, 120-124 (2011).
  8. Khalil, A. M. Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression. Proc. Natl. Acad. Sci. U.S.A. 106, 11667-11672 (2009).
  9. Zhao, J. Genome-wide identification of polycomb-associated RNAs by RIP-seq. Mol. Cell. 40, 939-953 (2010).
  10. Meller, V. H., Wu, K. H., Roman, G., Kuroda, M. I., Davis, R. L. roX1 RNA paints the X chromosome of male Drosophila and is regulated by the dosage compensation system. Cell. 88, 445-457 (1997).
  11. Franke, A., Baker, B. S. The rox1 and rox2 RNAs are essential components of the compensasome, which mediates dosage compensation in Drosophila. Mol. Cell. 4, 117-122 (1999).
  12. Gupta, R. A. Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature. 464, 1071-1076 (2010).
  13. Tsai, M. C. Long noncoding RNA as modular scaffold of histone modification complexes. Science. 329, 689-693 (2010).
  14. Zappulla, D. C., Cech, T. R. RNA as a flexible scaffold for proteins: yeast telomerase and beyond. Cold Spring Harb. Symp. Quant. Biol.. 71, 217-224 (2006).
  15. Nagano, T. The Air noncoding RNA epigenetically silences transcription by targeting G9a to chromatin. Science. 322, 1717-1720 (2008).
  16. Carter, D., Chakalova, L., Osborne, C. S., Dai, Y. F., Fraser, P. Long-range chromatin regulatory interactions in vivo. Nature. 32, 623-626 (2002).
  17. Chu, C., Qu, K., Zhong, F. L., Artandi, S. E., Chang, H. Y. Genomic Maps of Long Noncoding RNA Occupancy Reveal Principles of RNA-Chromatin Interactions. Mol. Cell. (2011).
  18. Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A., Tyagi, S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat. Methods. 5, 877-879 (2008).

Comments

84 Comments

  1. hi,i has a great interest on CHIRP and your works are highly appreciated,but i want to know that where can i find the buffer list?thank you

    Reply
    Posted by: tang n.
    May 5, 2012 - 4:16 AM
  2. Hi there, thanks for the interest in our paper. As you pointed out the original article lacked the buffer list, which we have uploaded a while ago. Hope it helps!

    Reply
    Posted by: Ci C.
    December 18, 2012 - 5:20 PM
  3. Hello, I'm trying to perform ChIRP experiments and your protocol is very helpful to me.

    However, I have a question about the final concentration of biotinylated probes and the volume of the beads.
    My probes are 100µM so 1µL corresponds to 100pmol. I have 50 probes and my question is : should I use 100µL of magnetic C1 beads per 100pmol of probe, so in my case, 50 probes x 100µL = 5mL of beads ?

    Thanks a lot for the answer ?

    Reply
    Posted by: Sylvain F.
    October 31, 2012 - 4:47 AM
  4. I also have this question!

    Reply
    Posted by: Guihai f.
    November 28, 2012 - 7:51 AM
  5. Hi guys, thanks for the interest in our paper. That's indeed a common question I get, so apologize for not explaining that more clearly in the paper. The 100uM concentration refers to all probes, so if you have ² probes in the pool the individual concentration is 50uM per probe, and if you have 50 probes it's ²uM per probe. It follows that as you scale up number of probes in the pool you do not have to scale up the amount of beads to add. As a caution though, we have noticed that when I use too many probes the concentration of each probe drops too much and it actually adversely affects yield. Try not to use more than 50 probes if possible.

    Reply
    Posted by: Ci C.
    December 18, 2012 - 5:23 PM
  6. what is the fucntion of the Buffer kit ? I can not find it in the protocol.

    Thanks for your time!

    Reply
    Posted by: Guihai f.
    November 1, 2012 - 12:50 AM
  7. The buffer kit is simply a convenient collection of buffers provided by Ambion that includes 5M NaCl, 1M Tris, 0.5M EDTA, etc..

    Reply
    Posted by: Ci C.
    December 18, 2012 - 5:25 PM
  8. Hi, I refer to your paper in HOTAIR ChIRP. You used 48 oligo probes for this ncRNA. I am trying to do a HOTAIR ChIRP. I am wondering if the number of probes can be smaller to reduce cost without a huge compromise on specificity. Furthermore, I am not doing sequencing but a specific check on a genomic region of interest, so I supposed the background noise may be lower? Thank you!

    Reply
    Posted by: Yi Fang L.
    November 8, 2012 - 5:11 AM
  9. We share your desire to reduce number of probes used. In preliminary testing I've found that you can use as few as 1 probe per ²00 nucleotide of RNA target RNA. But that number has to be doubled considering if you need both "even" and "odd" set, so effectively tiling density is 1 probe / 100nt.

    Reply
    Posted by: Ci C.
    December 18, 2012 - 5:28 PM
  10. Hello,
    I enjoyed your paper very much.

    At the end of the video, and in the figure, you mention that in this process you can isolate RNA binding proteins that were associated with the RNA. My questions are: have you had success in doing this? What is the protocol for isolating the proteins? Do you just isolate the protein fraction from the Trizol, or is there a more specialized method? Is there a paper that may lay out use of this method?

    Thank you for your help.

    Reply
    Posted by: Gavin J.
    January 18, 2013 - 12:09 PM
  11. Hi,
    Great protocol. Just one question. You mention that all ChIRP steps have to be performed at 37C. I can imagine this is important for the hybridisation steps, but I was wondering why this was needed for the washing steps as well?

    Many thanks

    Reply
    Posted by: Sebastian V.
    March 15, 2013 - 10:21 AM
  12. The washing should be performed at 37C just like you would wash a northern blot at elevated temperatures. If this is logistically difficult for you, just warming up the buffer to 37C and washing on a room temp. shaker works well too.

    Reply
    Posted by: Ci C.
    March 15, 2013 - 1:47 PM
  13. Hello,
    Thanks for the detailed and very helpful protocol. Can you tell me what kind of DNA yields to expect from the pull down per input cell amount? I know it will depend on the lncRNA targeted, but I need an idea of what kind of amounts to expect. Could you tell me what you got for your HOTAIR hybridizations? My own hybs only result in a few nanograms of DNA, and I don't know how to determine whether that is nonspecific DNA binding to my beads or whether it is actual lncRNA-bound DNA? How do you control for this? Thank you for your help in advance.

    Reply
    Posted by: blake e.
    March 20, 2013 - 5:44 AM
  14. Hi,

    Congrats for setting up such a protocol. It might have been discussed already but I would like to have an idea about the typical amounts of DNA and RNA that one might expect from 20 x 10 ^6 cells. This of course is transcript specific, but should we expect nanograms, picograms for either DNA and RNA?

    Also have you tried FA instead of glutaraldehyde for x-linking? Many thanks for your time

    Reply
    Posted by: Antonis A.
    November 26, 2013 - 12:15 PM
  15. Hi,

    Thanks for the detailed protocol. I am about to embark on it over the next week or so and had a question regarding the sonication. You advise a Bioruptor for this step however we only have a Covaris available. Would that provide acceptable sonication and do you have any advice regarding appropriate settings?

    Thank you

    Joshua

    Reply
    Posted by: joshua b.
    March 2, 2014 - 12:24 AM
  16. Yes Covaris works, we've found that it generally reduces sonication time by 30-50% compared with Diagenode. Actual time and intensity needs to be optimized for each machine and cell line.

    Reply
    Posted by: Ci C.
    March 3, 2014 - 5:51 PM
  17. Hi,

    Thanks for the detailed protocol. I am about to embark on it over the next week or so and had a question regarding the sonication. You advise a Bioruptor for this step however we only have a Covaris available. Would that provide acceptable sonication and do you have any advice regarding appropriate settings?

    Thank you

    Joshua

    Reply
    Posted by: joshua b.
    March 2, 2014 - 6:32 AM
  18. To all interested ChIRPers, hybridization can be performed overnight instead of 4hours. Doing so greatly reduces hands-on time. Happy ChIRPing!

    Reply
    Posted by: Ci C.
    March 3, 2014 - 8:14 PM
  19. Hi Ci,

    I have one more issue to resolve regarding ChIRPseq that I would appreciate your input on please (or other ChIRPers that have had similar problems).

    We have been unable to produce lysates using glutaraldehyde despite following the protocol to the letter and getting the Sigma brand that is recommended. Even before sonication, the DNA is degraded to a thick band below 200bp. When we try exactly the same process but substitute 1% formaldehyde (which we normally use for ChIP and 3C) or even plain PBS, the DNA is fine. We have even tried doing everything on ice and reducing the centrifuge steps to only 500g but to no avail.

    My questions therefore are:

    1/ Has your team had similar issues with glutaraldehyde in the past or found it to behave quite differently to formaldehyde (apart from needed longer sonication and harder centrifuging)?
    2/ Though you have shown glutaraldehyde to give a better signal to noise ratio, could we still expect to get usable data using 1% formaldehyde or is it not worth the time and effort?

    Thank you for your time

    Joshua Betts

    Reply
    Posted by: joshua b.
    April 16, 2014 - 8:07 AM
  20. Dear Joshua,

    Thanks for your interest in our protocol. It's unfortunate to hear that you're experiencing DNA degradation. We've never experienced this issue, nor have we heard similar problems from our collaborators who successfully did ChIRP. Usually we're more concerned with RNA integrity, naturally, and if you're careful that's not a problem either.

    Could you tell me how you're extracting DNA, especially prior to sonication?

    Regarding alternative crosslinking methods, for certain lncRNAs we've found that strong formaldehyde crosslinking also works (3% for 30min is a good starting point). 1% formaldehyde usually does not work.

    Best,
    Chu

    Reply
    Posted by: Ci C.
    April 16, 2014 - 1:55 PM
  21. Dear Chu,

    Thank you for your advice - I will try the protocol again but substitute 3% formaldehyde for the glutaraldehyde. Does that require any other adjustments apart from slower centrifuge spins and shorter sonication times?

    In regards to my DNA isolation, I lysed the cells as per the protocol and then treated with proteinase K for 45mins (at 50deg or 65deg - either seemed ok). I then RNAse treated the sample and either phenol chloroform extracted the DNA or instead used the Qiagen PCR columns before running the product on a 1% agarose gel. The columns gave more of a smear on the gel, whilst the phenol chloroform extraction just had a broad band below 100bp.

    When I performed exactly the same procedures on formaldehyde treated cells, I obtained a bright band of gDNA at the top of the gel as well as a more faint diffuse band below 100bp. This is what we normally see when working with formaldehyde. Am I right to expect a strong band of genomic DNA on the gel when using glutaraldehyde before sonication, or does the glutaraldehyde break the gDNA down during the fixation process? Does your glutaraldehyde fixed sample normally start as a broad smear even prior to sonication?

    Our group has a lot of experience with ChIPseq, 3C and 4Cseq library preparation but we have never dealt with glutaraldehyde before so are unsure what to expect.

    Any further suggestions would be greatly appreciated.

    Thanks

    Joshua

    Reply
    Posted by: joshua b.
    April 24, 2014 - 9:26 AM
  22. Before preparing your ChIRP library for sequencing, do you check your DNA size distribution (for example using Agilent Bioanalyzer). If so, do you see a similar distribution to that seen for a typical ChIP sample (200-800 bp range)?

    Reply
    Posted by: Eric S.
    August 29, 2014 - 2:43 PM
  23. Dear Chu
    I have two questions:
    1.In designing your chirp oligos have you tried to reduce the spacer between biotin-oligonucleotides. Is there any steric reason for choosing 1 probe / 100nt?
    2. Did you tried to crosslink adherent cells directly on plate? we have problem in properly resuspend our differentiated murine myoblasts in PBS+glutaraldehyde.

    thanks in advance
    regards

    Mariangela

    Reply
    Posted by: Mariangela M.
    October 16, 2014 - 9:56 AM
  24. Dear Mariangela,

    1. It's both a cost consideration (fewer probes are cheaper) and also a biochemical noise consideration (more probes introduce higher chances for probe:DNA direct hybridization).

    2. Yes we've found no difference fixing on plate vs. in suspension. Feel free to pick and choose what works best for you.

    Best,
    Chu

    Reply
    Posted by: Ci C.
    October 17, 2014 - 10:43 PM
  25. Dear ChIRP community,

    We are having problems getting ChiRP peaks with our transcripts. We have tried ChiRPing against 4 different transcripts and for all of them we were able to get very good fold enrichments in the genomic locus of each transcript (on average 50 to 200 x over background) capturing native transcription but we cannot find other binding sites in the genome for any of them. We have good reasons to believe that some or all of our transcripts are chromatin localized and we also know that the signal in the genomic loci is RNA dependent because it is dramatically reduced when we ChiRP after knocking down our transcripts of interest.

    Our transcripts are low in expression, on average 3 to 10 copies per cell. Any ideas as to how can we improve/solve the issue? Is it abundance that is limiting the system? Has anyone experienced/solved something similar?
    We tried the normal protocol, and now we are trying with 3 % FA in parallel to glut. Any suggestions would be very welcomed!

    Antonis

    Reply
    Posted by: Antonis A.
    November 13, 2014 - 5:18 AM
  26. Hi Antonis, I'm afraid the copy number is simply too low. In all of the ChIRP paper published so far I don't think anyone went that low.

    Your enrichment of the genomic loci could be complicated by ChIRP probes pulling down the DNA directly. Do an RNase-control to see if the signal is still there.

    Reply
    Posted by: Ci C.
    November 13, 2014 - 8:52 AM
  27. Dear Chu,

    Thank you for the prompt reply-yes I know that with such expression levels, ChIRP is challenging. I can try the RNase control but as mentioned I am confident about the RNA-dependence of the signal, because it falls back to LacZ levels when I knock down the transcripts. I guess we will have to try increasing the sensitivity of the experiment. Could an increase in x-linking time help here, have you guys tried 20 min of glut x-linking?

    Reply
    Posted by: Antonis A.
    November 18, 2014 - 3:28 AM
  28. Hello,
    I enjoyed your paper very much.

    At the end of the video, and in the figure, you mention that in this process you can isolate RNA binding proteins that were associated with the RNA. My questions are: have you had success in doing this? What is the protocol for isolating the proteins? Do you just isolate the protein fraction from the Trizol, or is there a more specialized method? Is there a paper that may lay out use of this method?

    Thank you for your help.

    Reply
    Posted by: jason h.
    November 26, 2014 - 10:15 PM
  29. Dear ChIRPers, we're happy to announce that you can now officially design and order ChIRP from Biosearch Technologies (Single molecule fish). Scientists developing ChIRP probes using the Stellaris probe designer and following the guidelines from our publication can now email Sheila Semaan at Biosearch Technologies, Inc. for help with ordering the probes in the proper format. Sheila’s contact info is as follows:

    Sheila J. Semaan, Ph.D., Associate Product Manager
    Biosearch Technologies, Inc.
    Email: ssemaan@biosearchtech.com

    Reply
    Posted by: Ci C.
    December 17, 2014 - 9:33 AM
  30. Dear ChIRP community,

    You can soon study RNA-protein interactions in vivo using ChIRP-mass spec. The story is coming out in Cell on April 2nd. Stay tuned!

    Reply
    Posted by: Ci C.
    March 25, 2015 - 5:42 AM
  31. Hello, thank you for the protocol! I also want to perform a RNA pulldown using your protocol and i am very excited for your RNA-protein interaction protocol! I have one question regarding the biotin oligos. Do they need to be 3' biotinylated or can they also be 5' biotinylated. Have you tested this? And you say nothing about (salt, HPLC) purification of the oligos. Thanks for answering my questions.

    Reply
    Posted by: Christine C.
    March 30, 2015 - 10:01 AM
  32. and is it necessary to order Biotin-TEG oligos or is "standard" biotin sufficient?

    Reply
    Posted by: Christine C.
    March 30, 2015 - 11:33 AM
  33. Hi Christine, thanks for your interest in our technique. Regarding your questions:

    1) 3' biotinylation is preferred, because oligos are synthesized 3' to 5'. This way, we ensure all oligos start with a biotin tag. 5' biotin tags are usually added to a synthesized oligo, and that increases the chance of untagged probes, which is less than ideal. However if you can ensure high tagging efficiency on 5' end, and the cost is advantageous, I don't see why it won't work!

    2) We use a disposable reverse phase column called "Glen-Pak DNA purification cartridge," which is a very efficient and cost-effective system. These are really short DNA probes and HPLC or PAGE would be necessary (also probably can't tell tag from untag probes).

    3) TEG ensures no steric hindrance, I don't have hard evidence on this but I think it's necessary.

    Reply
    Posted by: Ci C.
    March 30, 2015 - 10:03 PM
  34. Thank you for your answers! As I am performing your protocol with the aim of an RNA-protein pulldown, i have some other questions regarding the protocol, if you don't mind.

    - in the protocol before washed your magnetic beads in unsupplemented lysis buffer and you blocked the magnetic beads using BSA and yeast RNA- in the latest protocol you don't block. Why is that?

    - ChIRP-MS requires 100 Mio cells, ChIRP-seq 20 Mio cells. If i want to perform ChIRP-MS (or western blot) do i need 100 pmol of probe for each 20 Mio cells --> 500 pmol of probe for ChIRP-MS? --> 500 µl beads(??)

    Reply
    Posted by: Christine C.
    April 8, 2015 - 8:12 AM
  35. Hi Christine,

    - We no longer block as there's no difference in results, and we prefer a more streamlined protocol. BSA would also potentially contaminate ms results.

    - yes scale up beads accordingly. We do 1ml beads per experiment routinely. It gets expensive, but hopefully you'd only have to do it once or twice, and validate with western in a smaller scale later. For western of more abundant chirp hits, I do from 1/5 of cell pellets, but for less abundant ones I still use full amount.

    Reply
    Posted by: Ci C.
    April 8, 2015 - 11:36 PM
  36. Dear ChIRPers,

    ChIRP mass spec has just come online today at Cell. We're very pleased to introduce a very robust and straight-forward method that's optimized to study lncRNA:protein interactions. Check out the story at:

    http://www.cell.com/cell/abstract/S0092-8674(15)00312-8

    Happy ChIRPing!

    Best,
    Chu

    Reply
    Posted by: Ci C.
    April 2, 2015 - 9:00 PM
  37. Dear ChIRPers,

    Now you can easily design and order ChIRP probes with the latest optimization from Biosearch Technologies, the company that invented Single Molecule FISH.

    https://www.biosearchtech.com/chirpdesigner/

    Best,
    Chu

    Reply
    Posted by: Ci C.
    April 2, 2015 - 10:24 PM
  38. Hi, i'm trying to perform ChIRP-western and i have some questions concerning the methods.

    1) for a control, i also want to see if my RNA is present: how can i get a small aliquot of post-ChIRP beads to perform RNA elution with this aliquot
    2) for the protein biotin elution buffer: resuspend beads in what volume?
    3) for ChIRP-MS lysates are incubated with 30µl beads - can i reuse them?

    thank you in advance!

    Reply
    Posted by: Carolin C.
    April 14, 2015 - 7:18 AM
  39. Hi Carolin,

    thank you for your interested in our method.

    1) yes that's what we we as well. Simply take a 1% or 10% aliquot of your post-wash beads, and extract RNA.

    2) that's dependent on your starting beads volume. But there's a practical constraint, you don't want to have so much volume that after addition of TCA will exceed the capacity of an eppendorf tube.

    3) I'm not sure if I understand your question. Reuse lysate or beads?

    Reply
    Posted by: Ci C.
    April 15, 2015 - 12:20 AM
  40. thank you for the answers!
    to 2) again: if my beads are 100µl starting volume - so i resuspend 2x in biotin elution buffer and pool. Is 2x100µl then enough? and then add 25% of 100%TCA (50µl)?
    to 3) my question is, if i reuse the beads in the hybridization step. and just add another 70 µl beads (=100µl beads)

    Reply
    Posted by: Carolin C.
    April 24, 2015 - 11:23 AM
  41. so what do you recommend for my previous questions 2) and 3)

    another question: how do you calculate the amount of RNA retrieved? I retrieve only 10% of my RNA of interest calculated as % of Input. Furthermore,when i took a sample of what hasn't bound (after hybridization and capture of the beads) i get only 15% (fraction of washing doesn't include much RNA). so where has my RNA gone? i repeated several times… degradation?

    Reply
    Posted by: Carolin C.
    May 20, 2015 - 5:13 AM
  42. Hi, Carolin
    I am also, using ChiRP. Can you tell me the equation you used to calculate the % of your retrieval

    thanks

    Reply
    Posted by: u.
    June 28, 2016 - 1:22 AM
  43. Hi Carolin, May I ask in the end with your 10-20% retrieval rate of RNA, can you identify new proteins by MS via UV crosslink? Thanks.

    Reply
    Posted by: Zhenqiu H.
    May 17, 2018 - 6:20 PM
  44. Dear ChIRPers, we're glad to introduce the Magna ChIRP kit from Millipore. It's a simple pre-made reagent set for scientists who prefer the reliability and user-friendliness of a reputable brand.

    https://www.emdmillipore.com/US/en/product/EZ--Magna-ChIRP-RNA-Interactome-Kit---Isolation-and-characterization-of-non-coding-RNA%3Achromatin-complexes,MM_NF-17-10495

    Happy ChIRPing!

    Best,
    Chu

    Reply
    Posted by: Ci C.
    April 15, 2015 - 12:35 AM
  45. Hi Chu

    Thanks for sharing this information.
    Did anyone try this kit already and how good is it? Kits are easy to use and save time for reagents.

    Reply
    Posted by: Liu Z.
    April 16, 2015 - 5:17 PM
  46. Dear Chu,

    Many thanks for your excellent protocol. I'm designing a probe set and have been looking at the probes you designed for HOTAIR and XIST for inspiration. I noticed that some of the probes designed for XIST target the sequence at multiple loci (e.g. Probe 10), albeit ocassionally with a single base mismatch. Either way, I assume this may reduce the number of probes needed. Could I ask whether this was a deliberate design strategy and, if so, does the Biosearchtech ChIRP designer incorporate this strategy?

    Thanks

    Reply
    Posted by: Sebastian V.
    April 16, 2015 - 11:31 AM
  47. Dear Chu

    I feel exciting when I am reading your chirp paper in molecular cell. I have 2 questions that need your input.

    1) Have you or others you know tried in vivo tissue samples and if so how it works? I realized that your molecular cell and the latest cell paper used cell lines primarily.

    2) If I have a non-coding RNA which is 400 bp alone, and cannot generate more than 8 probes based on the parameters provided by https://www.biosearchtech.com/chirpdesigner/ ? What should I do ? Could I just reduce the spacer length between odd and even probes ? do you think it will increase background due to the increased probe density?

    Thanks

    Zhiyong

    Reply
    Posted by: Liu Z.
    April 16, 2015 - 4:49 PM
  48. Hi Zhiyong,

    Thank you for your kind words about our paper. Regarding your questions:

    1) Yes we have ChIRPed in drosophila tissues with great success. The key is probably to have small enough chunks of tissue that can be crosslinked effectively and thoroughly.

    2) Email the Biosearchtech team and they can help you manually reduce the number of probes.

    Good luck!

    Best,
    Chu

    Reply
    Posted by: Ci C.
    May 22, 2015 - 11:15 PM
  49. hey there, i was able to retrieve RNA with your method, but i'm not retrieving RNA bound protein (even though i did see a milky pellet after TCA and acetone treatment). i cross link with UV but the other steps i perform are just the same you are doing.
    i'm not so sure about this competetive elution method by using free d-biotin - and do you no longer add protease inhibitors in your buffer? the beads are simply eluted by using biotin buffer, rotate sample for 20 min,RT and 10 min mixing at 65°C (x2). This is it? then everything should be in my buffer and i continue with TCA precipitation ON.
    sorry if i this sounds stupid to you. i'm just looking for mistakes i could have made...

    Reply
    Posted by: Carolin C.
    June 10, 2015 - 10:32 AM
  50. Hi Carolin, I'm glad you're able to get RNA yield, that's a very good sign. The reason that you're not getting proteins could be: 1) mass spec instrument not sensitive enough. The quality of your results varies A LOT with who runs your mass spec. We've had amazing experience with Bill Lane at Harvard. 2) UV cross linking is known to be super low yield. Unless your lncRNA is very abundant or you're prepared to start with a huge amount of material, I would suggest formaldehyde as your first step. 3) TCA and cold acetone with give you a tiny pellet no matter what (the detergent acts as a carrier that will precipitate by itself even in the absence of proteins). So unless you see a sizable pellet, the presence of a pellet itself doesn't mean anything. d-biotin elution is complete, assuming you're using the C1 beads and biotin from invitrogen. Protease inhibitors are absent at the elution step, because your solution should be quite pure and free of contaminating proteases at that point assuming good lab practice. Hope this helps!

    Reply
    Posted by: Ci C.
    June 10, 2015 - 5:45 PM
  51. hey ci! thank you for your answers!
    1) i'm doing western blot as a start, later on i will do mass spec - so thank you for your tip&hint! as i already know a 100% candidate to get retrieved with my (cytoplasmic) mRNA, i stained for this candidate and got no result.
    2) you also mentioned uv-crosslinking in your first protocol, do you have a comment or any experience on the time of cross linking or amount of joules? so far i cross linked 150mj/cm^2 but I'm thinking to increase that. also i need to scale up my cells/beads/probe as you're saying. i do not know if formaldehyde cross linking works in my case, as i have cytosolic mrna.
    3) yes I'm using c1 dynabeads and d-biotin from invitrogen! thanks for the explanation, that helps!

    thank you very much for your quick answers, tips and hints!

    Reply
    Posted by: Carolin C.
    June 11, 2015 - 8:43 AM
  52. hey there, repeated the experiment - this time i used 200 Mio MEF cells - 10 ml lysate and 1 ml beads, 10µl probe. i also increased cross linking to 300 mj/cm^2 - still no proteins (known RNA binding protein) detectable via western blot. i confirmed RNA pulldown via qPCR after cross linking from a small fraction of beads.
    is sonication necessary?
    any troubleshooting ideas?

    Reply
    Posted by: Carolin C.
    June 17, 2015 - 11:38 AM
  53. I would start with formaldehyde first. UV is known to cross link very inefficiently, and certain residues of contact may not be amenable to photocrosslinking at all. Yes sonication is necessary to soluble the cell lysate.

    Reply
    Posted by: Ci C.
    June 19, 2015 - 2:37 PM
  54. i'm using your 25%TCA / Aceton precipitation method to precipitate my proteins. Have you also experienced a pH change (when adding laemmli it turns green/yellow) due to rest TCA? And i think i don't get my pellet resuspended - even after mixing and resuspending. Isn't boiling for 30 min, 95°C destroying the proteins?

    Posted by: Carolin C.
    July 10, 2015 - 7:30 AM
  55. Yes I have had similar experiences before: pH change usually can be prevented by more careful and thorough acetone washes (although need to care not to disturb pellet), and over drying will cause difficulty in resuspension. I never air dry for over a minute. Boiling is completely fine. Try using the LDS samples buffer / Bis-tris gel system from invitrogen, these work great on chirp samples. If you are still having issues with pH, you can manually adjust a little with a moderate alkaline buffer.

    Posted by: Ci C.
    July 10, 2015 - 10:59 PM
  56. Hi Carolin,

    I've the same problem as you had. I could only retrieve 10-15% of RNA when compare to input RNA. Could you please share how you sort this problem?

    Thank you in advance!

    Reply
    Posted by: Ganeshkumar A.
    July 23, 2015 - 4:25 AM
  57. Hi Ganeshkumar, unfortunately i didn't solve the problem… but i found more RNA of interest in my beads when i directly resuspended them in trizol (leaving out proteinase K treatment) - since i'm interested in my proteins bound to the RNA the proteinase K treatment step is of no importance to me. But so far i still couldn't retrieve my RNA bound proteins.

    Reply
    Posted by: Carolin C.
    July 23, 2015 - 5:19 AM
  58. Hi Carolin,

    Thanks for your immediate reply. I'll also avoid Proteinase K step and try again.
    I've one more question. We always do two step RT-qPCR. So, the amount of RNA that I retrieve isn't enough to perform a RT reaction. How do you do your RT-qPCR? This question maybe a bit stupid, but i really wanna sort out all the issues as early as possible.

    Thanks in advance.

    Reply
    Posted by: Ganeshkumar A.
    July 23, 2015 - 5:27 AM
  59. Hello,
    my RNA amount is also quite low, measured by Nanodrop (Once i measured with qubit and could't get anything). I'm using max. volume possible for RT reaction and adapt for input, not bound… Then i perform qPCR with my cDNA

    But i suppose you cannot skip proteinase K step when your lysate is formaldehyde treated, or you need to boil it first to reverse the crosslink. i diluted my beads in trizol when i had non-crosslinked samples to check for retrieval percentage

    Posted by: Carolin C.
    July 23, 2015 - 7:12 AM
  60. We always do a one-step qRT-PCR (i.e. each qPCR well runs it's own RT). This in my hands is much more sensitive and convenient as well (you don't have to measure RNA or cDNA, just load equal portions of each sample). We use Strategene Brilliant II SYBR reagents and Roche 480 machine. Let me know if this helps with yield detected.

    Posted by: Ci C.
    August 4, 2015 - 8:32 AM
  61. Hi Ci,

    I did gene specific cDNA synthesis with 10ng of RNA from all conditions and treated with RNase H and A (in excess) and then proceeded to qPCR. This worked good for me and I'm getting around 14-25 fold enrichment in my odd and even probe samples when compared to input.

    Thank you and Carolin for your suggestions.

    Posted by: Ganeshkumar A.
    August 4, 2015 - 10:43 AM
  62. Hello,
    i have not yet retrieved any RNA bound protein, so i'm thinking about what issues to sort out. i have to say i can retrieve my RNA of interest (but only max. 20% of input)

    i'm wondering about the protein amount in your formaldehyde cross linked, sonicated and hypotone lysed cell lysats. I experienced the protein amount (measured with Bradford) to be 5-8 fold lower compared to uncross linked or Uv cross linked cells treated with a NP40 lysis buffer.
    Why is that, and will it affect my experiments? Any ideas why the protein amount is so low?
    Thank you in advance!

    Reply
    Posted by: Christine C.
    July 23, 2015 - 5:47 AM
  63. Hi Christine, off the top of my head the two main reason for less protein detected upon cross linking is 1) insufficient sonication, and 2) insufficient reverse cross linking (i.e. boiling). Regarding 1), you have to titrate sonication time of your lysate, and pick the minimum sonication required to release all proteins and RNA of interest. To test this, precipitate the lysate post sonication for 10 min at max speed, and run qRTPCR against your RNA of interest and western against the protein on both the supernatant and pellet, the latter resuspended in appropriate buffer. This way you'll know if there's anything left to be solubilized. 3% cross linked lysates can take significantly longer to solublized than your typical ChIP cross linking.

    Regarding 2), do no less than 30min boiling in sample buffer. I've titrate that also, and 10min or 20min recovers much less protein.

    Let me know if these help and let's start from here. Good luck!

    Reply
    Posted by: Ci C.
    August 4, 2015 - 8:29 AM
  64. Hello,
    Many thanks again for this superb protocol. I'm currently trying to optimise this for mRNA pull-down from fixed cells. I've tried various fixing conditions in parallel and dot-blotted for my RBP of interest vs a non-binding protein (GAPDH) after performing ChIRP. I found that 1% glutaraldehyde (10 min) samples blotted strongly for my RBP, but produced an equally strong signal for GAPDH. The latter was also the case for 3% formaldehyde (10 min), but blots of both GAPDH and my RBP weren't as strong. The only sample that showed a slightly stronger signal of the RBP vs GAPDH was 1% formaldehyde (10 min), but both signals were quite weak. All samples were sonicated for several 30 sec ON and 45 OFF cycles at maximum intensity before foaming to produce sheared RNA of <500 bp in optically clear samples.

    I tested the expression of mRNA of interest in samples pulled down with my tiling oligo set compared with a run using non-targeting scrabled oligos. A capture on unfixed cells produced a very high target yield in the tiling oligo sample and no mRNA in the scrambled control sample. However, performing the same pull down in the 1% formaldehyde-fixed+sonicated samples dramatically increased background (GAPDH mRNA) compared to the scrambled control. I'm curious if you found anything similar during the development of ChIRP and ChIRP-MS and if you might have any solutions? Would extra sonication perhaps help?

    Many thanks

    Sebastian Vencken PhD
    Post-Doctoral Researcher

    Reply
    Posted by: Sebastian V.
    September 13, 2015 - 8:06 PM
  65. Hi Sebastian,

    Have you had any luck adapting this technique to mRNA capture?

    Best,
    Mike

    Reply
    Posted by: Michael K.
    December 7, 2016 - 4:48 PM
  66. Mixed results to be honest. We went forward with 1% formaldehyde and tried different forms of sonication (incl. covaris which didn't give us effective fragmentation). Biased background was still an issue and the copurification of our RBP (Ago+miRNA) was much lower than expected. This has apparently successfully been performed before (https://www.ncbi.nlm.nih.gov/pubmed/23325846) but we didn't manage to reproduce these results for our target.

    Reply
    Posted by: Sebastian V.
    December 8, 2016 - 4:15 AM
  67. Thanks for the feedback. Does your miR-CATCH work well/better? Similar idea, no sonication, and probes attached to beads first. Have you ever tried to use this to look for bound proteins?

    I also ran across this mRNA pulldown from yeast (https://www.ncbi.nlm.nih.gov/pubmed/27641505). They also attached the oligos to the beads first, but the buffers look more like ChIRP.

    Reply
    Posted by: Michael K.
    December 8, 2016 - 11:44 AM
  68. We've had some pretty good results (and publications) from miR-CATCH, but have also discovered that the performance can be very dependent on both the target and oligo design.
    With thorough optimisation we managed to reduce background in miR-CATCH, but each target was a new challenge in this respect.

    We hoped that ChIRP would significantly reduce the issue of background, but we have found balancing fragmentation and signal to be difficult. Dot blots for Ago2 gave us very high background signal in scrambled controls when using gluteraldehyde, while 1% formaldehyde gave some better signal differentiation, but at reduced strength. If I'd take ChIRP further for mRNA:RBP(:miRNA) capture, I'd look at the sonication and fixing steps again as there may be further room for improvement. Unfortunately, I've moved to other things for now.

    Reply
    Posted by: Sebastian V.
    December 9, 2016 - 10:42 AM
  69. Hello!

    Many thanks for the detailed protocol, however i am having trouble at crosslinking and sonication steps! I am sorry if it sounds silly but I just want to identifiy what I am missing/doing wrong.

    In the near future, I want to scale up for ChIRP MS, however first want to get these steps right as CHIRP MS would consume 10 times the beads.

    Crosslinking - When I add Glycine to quench formaldehyde, it doesnt change color?

    Sonication - As used by your team, I am using Bioruptor to sonicate the Hela S3 lysate (20 Mi. cells/1ml supplemeted lysis buffer) in 15 ml falcon (Corning) with a probe attached to the cap. I cant seem to get a clear lysate as shown in your video. I have tried sonicating starting from 30 cycles to 300 cycles and it just froths towards the end. After centrifugation to remove cell debris, the lysate is still considerably cloudy.

    I see that a lot of people are working on optimising this method and would appreciate any tips / tricks that might help me get going.

    Looking forward to some suggestions.

    Best regards,
    Minakshi
    PhD Student

    Reply
    Posted by: Minakshi G.
    September 30, 2015 - 5:06 AM
  70. Hi Minakshi, thanks for your interest in our protocol. Re your questions:

    - glycine doesn't change the color of formaldehyde, just glutaraldehyde.
    - check our bioruptor to make sure power is normal and probes are centered correctly. It shouldn't take more than a couple hours even for hardy cell lines. Frosting is strange and indicates to me that you may not be centering the probe correctly and thus losing efficiency.

    Reply
    Posted by: Ci C.
    November 2, 2015 - 2:46 PM
  71. Hi,
    I would have a question regarding the usage of formamide. My concern is that once the bottle is opened and the
    formamide is exposed to oxygen, it will begin to oxidize to formic acid. Do you take any precautions to prevent oxidation? How do you store formamide for further use? Sigma suggests to purge formamide with nitrogen and store it frozen to prevent oxidation.

    Thanks a lot for your help.

    Best wishes,
    matjaz

    Reply
    Posted by: Matjaz B.
    November 2, 2015 - 4:53 AM
  72. Hi Matjaz, thank you for your interest in our technique. formamide is indeed subject to oxidation and ionization. Re-deionization is laborious, so we tried our best to protect the reagents by parafilming around the bottle cap after each use and store it at 4C. I usually run through a 1 liter bottle in a year with repeated opening and closing, and have not observed any effect on results over time. Hope this helps.

    Reply
    Posted by: Ci C.
    November 2, 2015 - 2:44 PM
  73. Hello,

    I have another technical question. Even though our probes are very specific (we detect target RNA using different probes with high specificity), we have problem with high DNA background signal. Have you also had a similar problem? One way to resolve the issue would be to pre-clear lysates with beads before the hybridisation step. Have you ever implemented this step? If yes, have you done the pre-clearing in lysis buffer with or without hybridisation buffer?

    Many thanks,

    - matjaz

    Reply
    Posted by: Matjaz B.
    November 3, 2015 - 7:36 AM
  74. Hi Ci,

    Thanks for providing such an excellent protocol. I have a question about probe design. The 3' half of lncRNA I interested in (5kb in length) was fully cover by repeat elements. Because ChIRP probe Designer ignored the repeat sequence when designing probe, I found almost no probe target to the 3' half of this lncRNA. I'm afraid that I will miss all proteins bind to the 3' end of lncRNA if I only use the probe target to the 5' end of lncRNA. I wonder how do you handle lncRNA contains large number of repeat sequence?

    Thanks for any help.

    Sincerely,
    Jian-You

    Reply
    Posted by: Jian-You L.
    December 3, 2015 - 9:48 PM
  75. Hi all!

    Thank you for sharing this protocol!
    I have just analysed the RT-qPCR results coming from the RNA samples in order to analyse the lncRNA retrieval and I have retrieved a very small percentage of the lncRNA. From the input and the ChIRP samples I have used the same volume of sample (for the qPCR, around 36ng for the input and 21g for the ChIRP) and I have obtained a Ct of 30 for the input and 37 for the ChIRP measuring my lncRNA of interest. How do you normalise the results? Do you use the same volume or ng of the different RNA samples? Do you use a normalisation method similar to the ChIP one? https://www.thermofisher.com/uk/en/home/life-science/epigenetics-noncoding-rna-research/chromatin-remodeling/chromatin-immunoprecipitation-chip/chip-analysis.html

    Thank you in advance

    Isabel

    Reply
    Posted by: Isabel R.
    December 17, 2015 - 9:30 AM
  76. Hi, Isabel
    we experience along time to optimize the protocol in our lab around 9 month for lncRNA and we found the result unstable for different lncRNAs but for your normalization we usually use a normalized methods like Chip experiment for both GAPDH and our target,
    Hope that helps
    for more contact amr@mail.ustc.edu.cn

    Reply
    Posted by: u.
    December 8, 2016 - 7:17 PM
  77. Dear Chu:
    Recently I'm trying to perform ChIRP experiments and your detailed protocol is very helpful. However, I have some questions about cell lysis and sonication.
    1. I find that the supplemented lysis buffer cannot lyse the crosslinked cells. The crosslinked cells are only resuspended, after a few minutes they will sink to the bottom of the tube, is it normal?
    2. You emphasize that lysis buffer should be added fresh Protease Inhibitor, PMSF and Superase-in then proceed immediately to sonication after resuspended smooth, however, a sample will be added appropriate supplemented lysis buffer in proportion then devided into several parts to sonicate by limiting ultrasound equipment, and will it be affected?
    3. I only have a Covaris available for sonication in lab, I tried many times but could not find an appropriate setting. My DNA size distribution is normally a bright band of gDNA at the top, a bright band above 2000bp and a diffuse band from 2000bp to 100bp, if I continued to sonicate, the diffuse band would below 100bp, and there were considerably cloudy after centrifuged the sonicated samples. I want to know what and how much influence if DNA smear is less than 100bp or more than 500 bp? Do you have any advice regarding it?
    Looking forward to some suggestions, thank you in advance!
    Best regards
    mixue
    Ph.D. Student

    Reply
    Posted by: he j.
    July 17, 2016 - 3:37 AM
  78. Dear Dr CHU.
    First of all, sorry for my english, but I'm french.
    I work on lncRNA and I really appreciate your two articles on ChIRP and Chirp-MS, and since some months I try to set it up, but unfortunately without result until now.
    => I designed the probes as you know but my lncRNA is about 2.3kb so i was able to design only 26 probes. I also ordered your Positive and Negative Control.
    => I amplified the cells until 200 to 300 million and I cross linked them with 3% formaldehyde 30min and stop it with Glycine 0.125M 5 to 10 min, as you mention.
    => My sonication was tested in order to have a smear of DNA between 100 and 600bp and it took between 120 and 140min. I use a Bioruptore in cold room and I have to change water and ice every 15min in order to maintain the low temperature. Samples are centrifuged and debris are removed.
    => I use 2 to 3 aliquots of 1mL of lysed cells for each test, and the indicated quantity of probes. Of course before incubation, samples are pre-incubated with prewashed beads.
    => First incubation is done at 37 ° C in a shaking system, overnight. Second incubation (after addition of the prewashed beads) is done at 37 ° C for 1 hour.
    => Wash is done 5 times with wash buffer, then samples are eluted (using elution buffer freshly prepared with Sigma-Aldrich Biotine) and precipitated with TCA and Aceton. Pellet are directly dissolved in Laemmli, incubated 30min at 95°C and loaded with 12% acrylamide gel. After 1h at 200V the gel is stained with Coomassie Blue and destained 3 times 45min.
    I found NO BANDS even with Positive and negative controls, but after elution, when I checked the presence of my lncRNA in the sample selected with my probes, the result is positive.

    Could you tell me what are the possible mistakes that I could do ....?
    Thanks a lot.

    Reply
    Posted by: Sebastien C.
    January 3, 2017 - 5:02 AM
  79. Dear Dr. Chu,

    Good day! I am Anchilie, a PhD student, and I am really interested in applying your discovered technique in plants. I am currently doing the ChiRP-MS in Arabidopsis but I don't seem to get any RNA-bound proteins in my sample.

    I would just like to briefly tell you how I do the ChiRP-MS in plants. First, I harvest 3g of 14-day old seedlings and crosslinked it with 1% formaldehyde. From these, I then do nuclear extraction and do sonication. From the 1mL sonicated chromatin, I dilute it with 2mL of hybridization buffer, add 1ul probe and 100ul of beads and incubated then in 37C for O/N or 4 hours at 37C. From then I wash the beads 5x and precipitated the proteins using the biotin elution buffer and TCA method. The samples in 1x Laemmli buffer were submitted to LC/MS. I also check the enrichment in the qPCR and are enriched by 500-fold. However, upon analyzing my samples, the mass spec results only detected mitochondrial and chloroplastic proteins in the control and non-coding RNA

    I also tried many things like using 3% formaldehyde and UV crosslinking. I also did direct boiling of beads in Laemmli buffer. I also tried increasing the amount of material 10x accompanied with increasing the probe and the beads.
    But it doesn't really help.

    I would really appreciate to receive some feedback from you and your group with this matter.

    Best regards,
    Anchilie

    Reply
    Posted by: Anchilie M.
    February 27, 2017 - 4:49 AM
  80. Dear Anchilie,

    I am doing CHIRP in arabidopsis, you are the only only doing CHIRP as far as I know. I met some problems. I couldn't enrich my non coding RNA. So I want to know how did you do your CHIRP? Could you please share your protocol with me? Thanks!

    Reply
    Posted by: Zhi-Hong L.
    June 19, 2017 - 7:56 AM
  81. Hi guys, can anyone help me how the percentage calculation for lncRNA retrieval is done.

    Thank you

    Reply
    Posted by: Sunil B.
    June 22, 2017 - 3:58 PM
  82. Hi,

    Thank you for sharing this protocol, I have a question. If I want to study the potential interaction between a protein and a long non coding RNA in rDNA locus (or eventually with the rDNA directly), do you think that your kit will work ?
    Thank you for your answer

    Reply
    Posted by: Burette M.
    April 12, 2018 - 3:27 AM
  83. Dear professor,
    Thank you for sharing this protocol! It helps me a lot. I have a question to ask. Since ultrasound may break the hydrogen bond, I want to ask whether it is necessary to perform sonication when I study weak RNA-RNA interaction, for example: lncRNA-miRNA interaction?

    Reply
    Posted by: Zihao L.
    May 26, 2018 - 10:52 PM
  84. Dear professor,

    Any idea if using a probe-based sonication (like the Branson sonifier) would work? Anyone has tried it ?
    in the previous lab, we were using the bioruptor however we don't have it where I am now unfortunately and needed help to optimize it on the Branson or probe-based sonifiers?

    Thanks

    Reply
    Posted by: Anonymous
    March 1, 2020 - 4:26 AM

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