GSM5124498: ucMSC-RNA-seq-Young-rep2; Homo sapiens; RNA-Seq
Total RNA was extracted from PD12 and PD32 hMSCs using the RNEasy Mini Kit (Qiagen 74104) following the manufacturer's protocol, including the optional DNase treatment. 100ng of total RNA was depleted of rRNA (NEB, E6310) following the manufacturer's protocol;NSCs were isolated by FACS using the surface markers prominin-1 (CD133) and the EGF receptor, and GFAP-GFP fluorescence. For sorting, cells were stained with 1:300 EGF-Alexa 647 (Molecular probes, E-35351), and 1:400 Prominin-1-biotin (eBioscience, 13-1331-80). Dead cells were excluded using propidium iodide and cells were sorted on a BD FACS Aria into PBS. aNSCs and qNSCs can be distinguished by the surface expression of EGFR. Using this scheme, aNSC are prominin-1+; GFP+; EGFR+ and qNSC are prominin-1+; GFP+; EGFR-. We performed this experiment in biological triplicate, generating each RNA-seq library from a single animal, using ~400 cells per library. hMSC rRNA-depleted RNA was used as the template for a stranded RNA-seq library using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB, E7760) following the manufacturer's protocol. mNSC RNA-seq libraries were generated by GENEWIZ LLC. using the SMART-Seq v4 Ultra Low Input RNA Kit for cDNA synthesis and the Nextera XT DNA Library Preparation Kit.