GSM3415970: DEK-rep1; Mus musculus; RNA-Seq
The fixed muscle stem cell isolation was following the isolation protocol of satellite cells, as described previously in Liu et al., 2015, except that Collagenase II for first digestion was used at 2000 U/mL.Cell suspension was sorted using a BD Influx cell sorter equipped with 405-nm, 488-nm, 561-nm and 633-nm lasers. The machine was carefully optimized for purity and viability. To get high quality RNA from fixed cells, the total RNA from fixed muscle stem cells were isolated using a miRNeasy FFPE Kit (Qiagen) following the protocol described in Thomsen et al., 2015. cDNA was generated following the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech) protocol. The RNA-Seq libraries were sheared with a Covaris S220 and generated using Ovation Ultralow DR Multiplex System 9-16 (NuGEN Technologies). The libraries were sequenced using Illumina NextSeq 500 (2 x 75).