# characteristics.description[].text
Protocols: Hb9::gfp mES line (Lee et al., 2004) was cultured on MEF feeders using standard embryonic stem cell culture techniques. To generate spinal cord neurospheres, mESCs were dissociated with trypsin and 10^6 cells were plated in suspension in 10cm low-attachment dishes in ADFNK media [ADFNK media [Advanced D-MEM/F-12 (Life Technologies): Neurobasal medium (Life Technologies) (1:1), 10% Knockout Serum Replacement (Life Technologies), 200 mM L-Glutamine (Life Technologies), and 0.1 mM b-mercaptoethanol (Sigma)]. After two days, generated embryoid bodies were split onto three new 10-cm plates with fresh ADFNK media supplemented with 1 μM all-trans retinoic acid (RA, Sigma) and 100 nM smoothened agonist (SAG, Calbiochem). Two days later, freshly supplemented media was exchanged (Wichterle and Peljto, 2008). On day 6, neurospheres containing Hb9::gfp+ motor neurons were dissociated with papain (Worthington Biochemical), passed through a 40μm filter, and the resulting single cell suspension was used for fluorescence activated cell sorting. Cells were sorted on a Becton Dickinson FACS Vantage SE DiVa using Coherent Sapphire 488nm solid state lasers (200mW) and collected directly into Trizol LS. RNA was isolated according to Trizol LS protocol. mESCs were transfected with Lipofectamine 2000 with two pX330 vectors (containing Cas9 and two individual guide RNAs) and a plasmid expressing a puromycin resistance cassette. Guide RNAs were chosen to create specific multiplexed deletions between the selected gRNAs. 18 hours after transfection, mESCs were treated with puromycin for 48 hours to select for transfected cells. mESCs were split onto 10cm dishes with low density to allow for single colony selection. Colonies were manually picked after 5-8 days, manually dissociated, and split onto a feeder free gelatin coated plate for genotyping and MEF feeders for continued culture and freezing. Colonies were screened by PCR for wild type and knockout mutations to identify homozygous deletions of the indicated genomic regions. Selected mES lines that passed two rounds of PCR screening were expanded. mRNA sequencing libraries were prepared using the TruSeq RNA Library Preparation Kit (v2) according to the manufacturer’s instructions (Illumina). Briefly, RNA with polyA+ tails was selected using oligo-dT beads. mRNA was then fragmented and reverse-transcribed into cDNA. cDNA was end-repaired, index adapter-ligated and PCR amplified. AMPure XP beads (Beckman Coulter) were used to purify nucleic acids after each step. 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.