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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