शाही सेना शोधन द्वारा chromatin अलगाव (कूजन)
Summary
कूजन लंबी noncoding RNAs के जीनोमिक बाध्यकारी साइटों (lncRNAs) नक्शे के लिए एक उपन्यास और तेजी से तकनीक है. विधि विरोधी भावना खपरैल का छत के oligonucleotides विशिष्टता lncRNA बाध्य जीनोमिक साइटों की गणना की अनुमति का लाभ लेता है.
Abstract
Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA’s structure or functional domains.
Protocol
Discussion
यहाँ हम कूजन seq, vivo में lncRNA बाध्यकारी जीनोम चौड़ा साइटों में मानचित्रण का एक विधि का वर्णन किया. सफलता के लिए कुंजी पैरामीटर oligonucleotide जांच और glutaraldehyde crosslinking खपरैल का छत के विभाजन पूल हैं. संबंध जांच की डिजाइन शाही सेना अनुक्रम दिया सरल है और शाही सेना की संरचना या कार्यात्मक डोमेन का कोई पूर्व ज्ञान की आवश्यकता है. दो प्रजातियों में तीन नहीं बल्कि विभिन्न RNAs – roX2, TERC, और HOTAIR साथ हमारी सफलता से पता चलता है कि कूजन seq संभावना कई lncRNAs generalizable है. सभी प्रयोगों के साथ के रूप में, देखभाल और उचित नियंत्रण करने के लिए परिणामों की व्याख्या करने के लिए आवश्यक हैं. विभिन्न lncRNA शर्तों के अनुमापन, और अलग आत्मीयता जांच या crosslinkers के चयन के रूप में इस तरह की स्थितियों का विवेकपूर्ण परिवर्तन की आवश्यकता हो सकती है, शाही सेना chromatin के बातचीत के विभिन्न पहलुओं पर प्रकाश डाला सकता है. चिप seq तरह, सभी बाध्यकारी नहीं घटनाओं जरूरी कार्य कर रहे हैं, और अतिरिक्त अध्ययन के लिए शाही सेना ओ के जैविक परिणाम का पता लगाने के लिए आवश्यक हैंchromatin पर ccupancy. बहरहाल, हम अन्य chromatin से जुड़े lncRNAs, जो अब 8,9 हजारों में संख्या के शोधकर्ताओं के लिए इस तकनीक के कई दिलचस्प आवेदनों की उम्मीद है. बस के रूप में चिप seq डीएनए प्रोटीन बातचीत के जीनोम चौड़ा अन्वेषणों के लिए दरवाजा खोल दिया है, कूजन 'आरएनए interactome' की seq अध्ययन जीव विज्ञान के कई नए रास्ते प्रकट हो सकता है.
Disclosures
The authors have nothing to disclose.
Acknowledgements
हम टी. त्रिशंकु, एम सी धन्यवाद. Tsai, ओ मनोर, ई. सहगल, एम. कुरोडा, टी. Swigut, और विचार विमर्श के लिए मैं Shestopalov. विज्ञान, प्रौद्योगिकी और अनुसंधान (सीसी) सिंगापुर, R01 CA118750 एनआईएच और R01 HG004361 (HYC), और पुनर्योजी चिकित्सा HYC () के लिए और कैलिफोर्निया इंस्टीट्यूट की एजेंसी द्वारा समर्थित है. HYC हॉवर्ड ह्यूजेस मेडिकल इंस्टीट्यूट जल्दी कैरियर वैज्ञानिक है.
Materials
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:
| Name of the reagent | Company | Catalogue number |
| Glutaraldehyde (EM grade) | Sigma Aldrich | G5882-10x10ml |
| Motorized pellet mixer | VWR | V8185-904 |
| Protease inhibitor | Roche | 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 | 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 | J.T. 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 |
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