GSM2839875: MN_day7_r1; Mus musculus; RNA-Seq
Total RNA was extracted at days 0 (mouse embryonic stem cells), 2, 3, 4, 5, and 7 (motor neurons) of each differentiation using a Trizol/RNeasy hybrid protocol. Briefly, cells are homogeneized using a recommended volume of Trizol (Invitrogen), chloroform is added in the recommended volume and the mix is shaken vigorously. After centrifugation to allow phase separation, the aqueous phase is kept and mixed with 70% ethanol 1:1. The protocol follows using an RNeasy column (QIAGEN) and following the manufacture's instructions. For days 5 and 7 a step was added previous RNA extraction: cells were sorted using a BDFACSAria Cell Sorter (BD) to isolate only the GFP-positive fraction, therefore enriching the sample in motor neurons. GFP-positive cells were sorted into Trizol LS (Invitrogen) and extraction of RNA proceeded as before The RNA samples were first treated with DNase, then one library per sample was prepared using Illumina's TruSeq RNA Sample Prep Kit, where polyA-fragments were selected, followed by cDNA synthesis and ligation of amplification and sequencing adapters. Libraries were then individually barcoded and then pooled with 6 libraries per lane on an Illumina HiSeq 2500 instrument (Illumina).
GEO Accession
GSM2839875