GSE126534
Transcriptional profiling (RNA-seq) of wild-type and Arhgap35 knockout embryonic motor neurons (E12.5)
Ventral spinal cord regions micro-dissected from E12.5 Arhgap35 (aka, p190) knockout embryos or control littermates (wild-type or p190+/-) were pulled and dissociated with papain (Worthington Biochemical Cat# LK003153) according to the manufacturer's instructions. Cells were sorted on a Becton Dickinson FACS Vantage SE DiVa using Coherent Sapphire 488 nm solid state laser and collected directly into RLT lysis buffer containing ß-mercaptoethanol (Qiagen). RNA was isolated using the RNeasy Mini Kit (Qiagen) with on-column DNase digestion (Qiagen RNase-Free DNase Set). Each sample contained between 100,000 and 200,000 GFP+ motor neurons isolated from 2-5 spinal cords during a single experimental session. Five p190-/- and five control RNA samples were quantified with Agilent 2200 TapeStation system prior to preparation of mRNA-sequencing libraries (50 bp single-end) using the Illumina TruSeq RNA Library Preparation Kit (v2) according to the manufacturer's instructions. Libraries were sequenced with Illumina HiSeq 2500 platform. "Negative" samples are from FACS-sorted Hb9:GFP-negative cells. Overall design: FACS sorting and RNA-seq of motor neurons expressing Hb9:GFP reporter from either control or Arhgap35 knockout embryos (E12.5)