GSM5311406: Cd_50 [S180-2_S7]; Mus musculus; RNA-Seq
Whole brains were removed, flash frozen on dry ice, and kept at -80C until RNA was harvested using Qiagen AllPrep DNA/RNA/miRNA kit. Purification of messenger RNA (mRNA) was performed using oligo-dT beads provided in the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs [NEB]). Complementary DNA (cDNA) libraries for Illumina sequencing were constructed using the NEBNext Ultra Directional RNA Library Prep Kit (NEB) and NEBNext Multiplex Oligos for Illumina (NEB) using the manufacturer-specified protocol. Following adapter ligation, the samples were selected for a final library size (adapters included) of 400–550 bp using sequential AMPure XP bead isolation (Beckman Coulter). Library enrichment was performed and indexes for each sample were added during the protocol-specified PCR amplification. Amplified library fragments were purified and checked for quality and final concentration using an Agilent 2200 Tapestation (D1000 chip, Agilent Technologies) combined with a Qubit fluorometer (ThermoFisher). Libraries were pooled in equimolar amounts for clustering and sequencing. Illumina HiSeq 2500 DNA sequencer, utilizing a 125 bp single-end cycle sequencing kit.