title: GSM6004948: Abcd2-/- BV-2 rep2; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was extracted from 3 independent batches of BV-2 cells for each genotype (WT, Abcd1-/-, Abcd2-/-, Abcd1-/-/Abcd2-/-, and Acox1-/-) using RNeasy kit (Qiagen). RNA-Seq libraries were generated from 1000 ng of total RNA using TruSeq Stranded mRNA LT Sample Preparation Kit (Illumina, San Diego, CA). Briefly, following purification with poly-T oligo attached magnetic beads, the mRNA was fragmented using divalent cations at 94oC for 2 minutes. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. Strand specificity was achieved by replacing dTTP with dUTP during second strand cDNA synthesis using DNA Polymerase I and RNase H. Following addition of a single 'A' base and subsequent ligation of the adapter on double stranded cDNA fragments, the products were purified and enriched with PCR (30 sec at 98oC; [10 sec at 98oC, 30 sec at 60oC, 30 sec at 72oC] x 12 cycles; 5 min at 72oC) to create the cDNA library. 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.