RNA-Seq library preparation and sequencing: Site 1
Libraries were prepared using the Illumina (Illumina Inc. San Diego, CA, USA) TruSeq™ mRNA stranded kit as per the manufacturer’s instructions. 500 ng of total RNA was used for library preparation. ERCC synthetic spike-in 1 or 2 (Thermo Fisher Scientific Inc., Waltham, Massachusetts) was added to each total RNA sample and processed in parallel. The TruSeq method (high-throughput protocol) employs two rounds of poly-A based mRNA enrichment using oligo-dT magnetic beads followed by mRNA fragmentation (120-200 bp) using cations at high temperatures. First and second strand cDNA synthesis was performed followed by end repair of the blunt cDNA ends. One single “A” base was added at the 3’ end of the cDNA followed by ligation of the barcoded adapter unique to each sample. The adapter-ligated libraries were then enriched using PCR amplification. The amplified library was validated using a DNA tape on the Agilent 4200 TapeStation and quantified using a fluorescence-based method. The libraries were normalized and pooled and clustered on the HiSeq3000/4000 flow cell on the Illumina cBot. The clustered flowcell was sequenced on the Illumina HiSeq3000 system employing a single-end 101 cycles run, each sample was sequenced to an average depth of 25 M reads.