GSM2488462: RPI27_18; Mus musculus; RNA-Seq
Embryoid bodies were dissociated into single cells using the Worthington Biochemical Papain Dissociation System (LK003178). Single cell deposition was accomplished using a Beckman Coulter MoFlo Astrios EQ cell sorter into 96 well plates. Cells were then snap-frozen and subsequently lysed. Single-cell RNA-seq libraries were prepared using a modified CEL-seq protocol (Hashimshony et al., Cell Reports 2, 666-673 (2012)). Briefly, the mRNA in each cell lysate was reverse transcribed with barcoded primers, the single cell samples were pooled, second strand cDNA was synthesized, and then linearly amplified by in vitro transcription with T7 RNA polymerase. The amplified RNA (aRNA) was chemically fragmented and T4 RNA ligase was used to ligate Illumina 3’-RNA adapters. The aRNA was then run on an Agilent Bioanalyzer to assess proper fragmentation and then reverse transcribed to generate cDNA and subjected to PCR enrichment.