title: GSM2913129: V2a- lumbar rep 2; Mus musculus; RNA-Seq
library_construction_protocol: Cervical and lumbar segments were dissected in aCSF, and cell dissociation was conducted using Papain following the manufacturer's instruction (Worthington Biochemical). Via FACS (FACSDIVA or Influx, BD), cells were collected directly into Trizol (Life Technologies). 5-6 neonates at P0 were used from each litter, which typically yielded ~20K cells. ~100K Tom- cells were collected. 3 litters were used to obtain biological replicates. Total RNA was isolated and digested with TURBO DNase (Life Technologies). An Agilent Tape Station was used to determine RNA integrity (RIN) numbers prior to library preparation. Stranded mRNA-Seq libraries were prepared using the TruSeq Stranded mRNA Library Prep Kit according to the manufacturer's instructions (Illumina). Briefly, poly-A RNA was isolated using magnetic beads conjugated to poly-T oligos. mRNA was then fragmented and reverse-transcribed into cDNA. dUTPs were incorporated, followed by second strand cDNA synthesis. dUTP-incorporated second strand was not amplified. cDNA was then end-repaired, index adapter-ligated and PCR amplified. AMPure XP beads (Beckman Coulter) were used to purify nucleic acid after each steps of the library prep.