GSM1694664: control_rep2_RNA-seq; Homo sapiens; RNA-Seq
Day 28 MN cultures were dissociated into single cells using Accutase, and fixed in 4% paraformaldehyde (PFA). Monoclonal HB9 (MNR2) antibody (Developmental Studies Hybridoma Bank 81.5C10) was diluted 1:100 in blocking buffer (1% BSA) and 0.2% saponin (Sigma) in RNase-free PBS supplemented with 50 U/ml Superase.In (Ambion), and incubated with the cell suspension for 3 hours at room temperature. AlexaFluor-488 conjugated secondary antibody was diluted 1:1000 in blocking buffer, and incubated with the cell suspension for 45 minutes at room temperature. The cells were then washed with DPBS several times before FACS on BD FACS Aria. Sorted cells were treated with RNase-free Proteinase K for an hour at 50 °C, before addition of Trizol LS (Invitrogen) for RNA extraction. DNase treatment using Turbo DNA-free (Ambion) was performed to remove contaminating genomic. Illumina mRNA-seq libraries were prepared using the TruSeq RNA kit, using 200 ng of total RNA. RNA was differentially fragmented, DSN-Lite was used to remove rRNA-derived cDNA following reverse transcription (Adiconis et al., 2013). total DSN-lite RNA-seq, paired end seq
GEO Accession
GSM1694664