GSM2492620: NPAI-C1 (57532); Mus musculus; RNA-Seq
RNA was harvested using the RNEasy-Plus Kit (Qiagen;74136) and assessed for quality using the TapeStation (Agilent). Samples with RNA Integrity Number of 8 or greater were rRNA depleted using Ribo Minus (Invitrogen;K1550-04). rRNA-depleted samples were prepared using the TruSeq mRNA SamplePrep v2 kit (Illumina;RS-122-2001, RS-122-2002). The entire fraction of 0.1-3 ug of rRNA depleted total RNA was fragmented and copied into first-strand cDNA using reverse transcriptase and random primers. cDNA 3‘-ends were adenylated and adapters were ligated. One of the adapters had a 6-nucleotide barcode that was unique for each sample, allowing us to multiplex in a HiSeq flow cell (Illumina). Products were purified and enriched by PCR to create the final cDNA library. Libraries were checked for quality and quantity by TapeStation (Agilent) and qPCR using a library quantification kit for Illumina platforms (Kapa Biosystems;KK4835). They were clustered on the cBot (illumina) and all 9 samples were sequenced on each of 2 llanes, then the reads files for each sample were concatenated. Sequencing was 50-cycle, single end, on the Illumina HiSeq 2500 High-Output sequencer, using V4 chemistry