GSM1641279: RNAseq DE Sample 5: ERCC no1; Homo sapiens; RNA-Seq
Capture sequencing was performed by combining and modifying the NimbleGen SeqCap EZ Library SR User’s Guide V3.0 and the NimbleGen Arrays User’s Guide: Sequence Capture Array Delivery v3.2. RNA sequencing libraries of ribodepleted total RNA from three K562 biological replicates were created using the TruSeq® Stranded mRNA Sample Preparation Kit (Illumina). Different index adaptors (4,6,12) were added to each K562 biological replicate. Library input consisted of ribodepleted RNA from 5 μg original total RNA after quality checking. ERCC RNA Spike-In Control mix 1 (Invitrogen) was added to ribodepleted RNA to give a final ERCC concentration of 1% (replicate 1 and 2) and 1.08% (replicate 3). Library preparation was begun at the fragmentation step by the addition of 9 μl EPH buffer and the standard protocol followed until “Enrich DNA Fragments”. The 20 μl obtained at the end of the “Ligate Adaptors” step was increased to 21 μl with resuspension buffer and mixed well. One microliter of this solution was added to 19 μl resuspension buffer in a new 0.3ml PCR plate and mixed. This new plate was utilised for the “Enrich DNA Fragments” step to create amplified test libraries to guide PreCapture LMPCR with the remaining 20 μl. Precapture LMPCR and QIAquick PCR Purification Kit (Qiagen) were performed as described by the NimbleGen SeqCap EZ Library SR User’s Guide V3.0. To test LMPCR yields K562 biological replicate 3 libraries were amplified for 8, 9 or 10 cycles, quantified on the bioanalyzer and then pooled. K562 biological replicate 1 and 2 libraries were thus amplified for 9 cycles and the yield quantified by bioanalyzer. Capture hybridization was modified from the NimbleGen Arrays User’s Guide: Sequence Capture Array Delivery V3.2. The NimbleGen Hybridization System was set to 42°C and allowed 3 h to equilibrate. Equal nanograms of library from each K562 biological replicate were pooled, some of pooled library was allocated for pre-capture sequencing and 1ug of the pooled library utilized for capture. The library for capture was mixed with 300 µg human Cot-1 DNA (Invitrogen) and 3.34 µl 100 μm TS-INV-HE (hybridisation enhancing) Index Oligos (IDT) to each index adaptor plus 1 µl 1000 μm TS-HE Universal Oligo 1 (IDT) in a 1.5 ml tube. Lid of tube was pierced with an 18 gauge needle and the sample dried at 60 °C in a vacuum concentrator. Once dry the tube was given a new lid to prevent contamination through the needle hole. Library/Cot/Oligo mix was resuspended in 11.2 µl of nuclease and nucleic acid free water, vortexed for 10 s and centrifuged at max speed for 10 s. Sample was solubilised at 70 °C for 10 m before repeating vortexing and centrifugation. Nimblegen 2 x SC Hybridization Buffer (18.5 µl) and SC Hybridization Component A (7.3 µl) were added and vortexing and centrifugation repeated. Sample was denatured at 95°C for 10 m. During this time the “Prepare Mixers” procedure from the NimbleGen Arrays User’s Guide was performed with the omission of the compressed gas step. After denaturation samples was vortexed for 10 s, centrifuged at max speed for 10 s and incubated at 42°C until ready to load onto the NimbleGen Hybridization system. The ‘Load and Hybridize Samples’ procedure from the NimbleGen Arrays User’s Guide was performed to begin array hybridization. Hybridization was conducted for ~3 days. The NimbleGen Arrays User’s Guide protocol was followed to prepare the elution chamber, disassemble, wash and elute the captured DNA from the microarray, with the following modifications. Gasket and elution chamber was setup in a DNA-free laminar flowhood. All washes were conducted with 50 ml buffer in 50 ml falcon tubes. Minelute purification (Qiagen) was conducted using a microfuge. Captured DNA was recovered with two 30 µl elutions from a Minelute column and the final volume adjusted to 60 µl. Post-capture LMPCR and cleanup was performed similar to the NimbleGen SeqCap EZ Library SR User’s Guide V3.0, with the following modifications. LMPCR was run using 5x Phusion buffer for 17 cycles. Each PCR contained 70 µl of master mix and 30 µl of captured DNA. Quantity and quality of amplified captured DNA was determined by bioanalyzer. Enrichment of captured transcripts was measured by qPCR using SYBR Green PCR Master Mix and real time cyclers (Applied Biosystems). Enrichment was determined by the ratio of transcript abundance between the pre and post capture samples using equal nanograms of each. Enrichment was determined for three Nimblegen capture controls and 3 sequences captured specifically by the branchpoint array. Two transcripts not targeted by the capture array were also tested to examine the specificity of capture. Concentrations and volumes were as per the NimbleGen SeqCap EZ Library SR User’s Guide V3.0. Precapture and post capture samples (3 K562 biological replicates) were each sequenced on a single lane of an Illumina® HiSeq.
GEO Accession
GSM1641279