title: GSM8591166: O4, female, control, RNA, rep4; Mus musculus; RNA-Seq
library_construction_protocol: Brain O4+ oligodendrocytes were isolated by magnetic cell sorting (Miltenyi). RNA were purified using the Macherey Nagel RNA XS Plus kit. RNA-Seq libraries were generated according to manufacturer's instructions from 100 ng of total RNA using the Illumina Stranded mRNA Prep, Ligation kit and IDT for Illumina RNA UD Indexes Ligation (Illumina, San Diego, USA). Briefly, Oligo(dT) magnetic beads were used to purify and capture the mRNA molecules containing polyA tails.The purified mRNA were then fragmented at 94°C for 8 min and copied into first strand complementary DNA (cDNA) using reverse transcriptase and random primers. Second strand cDNA synthesis further generated blunt-ended double-stranded cDNA and incorporated dUTP in place of dTTP to achieve strand specificity by quenching the second strand during amplification. Following A-tailing of DNA fragments and ligation of pre-index anchors, PCR amplification was used to add indexes and primer sequences and to enrich DNA libraries (30 sec at 98oC; [10 sec at 98oC, 30 sec at 60oC, 30 sec at 72oC] x 13 cycles; 5 min at 72oC). Surplus PCR primers were further removed by purification using SPRIselect beads (Beckman-Coulter, Villepinte, France). The final libraries were checked for quality, quantified using capillary electrophoresis and sequenced on a Hiseq 4000 sequencer by the GENOMEAST platform.