Gerichte RNA Sequencing Assay Gene Expression en Genomic Wijzigingen karakteriseren

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Martin, D. P., Miya, J., Reeser, J. W., Roychowdhury, S. Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations. J. Vis. Exp. (114), e54090, doi:10.3791/54090 (2016).

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Abstract

RNA sequentie (RNAseq) is een veelzijdige werkwijze die kan worden gebruikt voor het detecteren en karakteriseren van genexpressie, mutaties, genfusies en niet-coderende RNA's. Standard RNAseq vereist 30-100000000 sequencing leest en kan meerdere RNA zoals mRNA en niet-coderende RNA's. We laten zien hoe gerichte RNAseq (capture) maakt een gerichte studie op geselecteerde RNA-producten met behulp van een desktop-sequencer. RNAseq capture kan een annotatie, lage, of tijdelijk tot expressie transcripten die anders kan worden gemist met behulp van traditionele methoden RNAseq karakteriseren. Hier beschrijven we de extractie van RNA uit cellijnen, ribosomaal RNA uitputting, cDNA synthese, het opstellen van barcode bibliotheken, hybridisatie en vastleggen van gerichte transcripties en multiplex sequencing op een desktop sequencer. We hebben ook een overzicht van de geautomatiseerde analyse pijpleiding, die kwaliteitscontrole assessment, uitlijning, fusion detectie, genexpressie kwantificering en identificatie van enkele Nuc omvatleotide varianten. Deze test maakt gerichte transcript sequentiebepaling genexpressie genfusies en mutaties te karakteriseren.

Materials

Name Company Catalog Number Comments
Thermomixer R Eppendorf 21516-166
Centrifuge 5417R Eppendorf 5417R
miRNeasy Mini Kit Qiagen 217004
Molecular Biology Grade Ethanol Sigma Aldrich E7023-6X500ML
Thermoblock 24 x 1.5 ml Eppendorf 21516-166
MiSeq Reagent Kit v2 (300-cycles) Illumina MS-102-2002
MiSeq Desktop Sequencer Illumina
PhiX Control v3 Illumina FC-110-3001
TruSeq Stranded Total RNA Kit with RiboZero Gold SetA Illumina RS-122-2301
25 rxn xGen® Universal Blocking Oligo - TS-p5 IDT 127040822
25 rxn xGen® Universal Blocking Oligo - TS-p7(6nt) IDT 127040823
25 rxn xGen® Universal Blocking Oligo - TS-p7(8nt) IDT 127040824
Agencourt® AMPure® XP - PCR Purification beads  Beckman-Coulter A63880
Dynabeads® M-270 Streptavidin Life Technologies 65305
COT Human DNA, Fluorometric Grade, 1 mg Roche Applied Science 05480647001
Qubit® Assay Tubes  Life Technologies Q32856
Qubit® dsDNA HS Assay Kit Life Technologies Q32851
SeqCap® EZ Hybridization and Wash Kits  (24 or 96 reactions) Roche NimbleGen  05634261001 or 05634253001 
Qubit® 2.0 Fluorometer  Life Technologies Q32866
10 x 2 ml IDTE pH 8.0 (1x TE Solution) IDT
Tween20 BioXtra Sigma P7949-500ML
Nuclease Free Water Life Technologies AM9937
C1000 Touch™ Thermal Cycler with 96–Well Fast Rection Module Biorad 185-1196
SeqCap EZ Hybridization and Wash Kits Roche Applied Science 05634253001
SuperScript II Reverse Transcription 200 U/μl Life Technologies 18064-014
D1000 ScreenTape Agilent Technol. Inc. 5067-5582
Agencourt RNAClean XP - 40 ml Beckman Coulter Inc A63987
RNA ScreenTape Agilent Technol. Inc. 5067-5576
RNA ScreenTape Ladder Agilent Technol. Inc. 5067-5578
RNA ScreenTape Sample Buffer Agilent Technol. Inc. 5067-5577
Sodium Hydroxide Sigma 72068-100ML
DynaBeads MyOne Streptavidin T1 Life Technologies 65602
DYNAMAG -96 SIDE EACH Life Technologies 12331D
Chloroform Sigma C2432-1L
KAPA HotStart ReadyMix KAPA Biosystems KK2602
NanoDrop 2000 Spectrophotometer Thermo Scientific
My Block Mini Dry Bath Benchmark BSH200
D1000 Reagents Agilent Technol. Inc. 5067- 5583
Vacufuge Plus Eppendorf 022829861 

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