GSM6927665: 19_9_18_HSC3 [JMCT17]; Mus musculus; RNA-Seq
RNA was extracted using the Macherey-Nagel™ Mini kit Nucleospin™ (Dutscher, # 872061) or Qiagen micro kit, depending on abundance of cell preparations, following the manufacturer's instructions. RNA concentration and purity were assessed using a Nanodrop One device (ThermoFisher Scientific) or a Qubit fluorometer (ThermoFisher Scientific) and a Qubit RNA hs assay kit (ThermoFisher Scientific), while RNA integrity was analyzed on a Bionanalyzer 2100 (Agilent). RNA preparations with RIN<6.0 were discarded. Total RNA-Seq libraries were generated from 38 to 100ng of total RNA using TruSeq Stranded Total RNA LT Sample Prep Kit with Ribo-Zero Gold (Illumina, San Diego, CA), according to manufacturer's instructions. Briefly, cytoplasmic and mitochondrial ribosomal RNA (rRNA) was removed using biotinylated, target-specific oligos combined with Ribo-Zero rRNA removal beads. Following purification, the depleted RNA was fragmented into small pieces using divalent cations at 94oC for 2 minutes. Cleaved RNA fragments were then copied into first strand cDNA using reverse transcriptase and random primers followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. Strand specificity was achieved by replacing dTTP with dUTP during second strand synthesis. The double stranded cDNA fragments were blunted using T4 DNA polymerase, Klenow DNA polymerase and T4 PNK. A single 'A' nucleotide was added to the 3' ends of the blunt DNA fragments using a Klenow fragment (3' to 5'exo minus) enzyme. The cDNA fragments were ligated to double stranded adapters using T4 DNA Ligase. The ligated products were enriched by PCR amplification (30 sec at 98oC; [10 sec at 98oC, 30 sec at 60oC, 30 sec at 72oC] x 12 cycles; 5 min at 72oC). Surplus PCR primers were further removed by purification using AMPure XP beads (Beckman-Coulter, Villepinte, France) and the final cDNA libraries were checked for quality and quantified using capillary electrophoresis. RNA preparations were first depleted from unwanted, abundant transcripts using Ribo-Zero Plus rRNA depletion kit (Illumina, # 20040526). cDNA synthesis, 3'end adenylation, adapter ligation and PCR amplification were performed using the TruSeq Stranded total RNA sample preparation kit (Illumina). DNAs were sequenced using a Hiseq4000 sequencer with 50b single reads. Sequencing depth was 75 million reads on average.