title: GSM8443046: MSC-iPS, day 18, replicate 2, sci-RNA-seq; Homo sapiens; OTHER
library_construction_protocol: For cell isolation for sci-RNA-seq or 10x sc-RNA-seq, whole embryoid bodies were collected, incubated in 2mg/ml Collagenase B in IMDM for 2 hours with trituration every 30min, followed by incubation in 2ml Gentle Cell dissociation reagent for 15min, followed by rapid trituration for manual dissociation, followed by filtration through a 70μM filter and resuspension in 3% FBS in PBS. For sci-RNA-seq, cells were then resuspended in a cell lysis buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% IGEPAL CA-630) followed by gentle pipetting, followed by filtration of the resulting nuclei through a 70μM strainer and incubation in 4% paraformaldehyded in 1.25X PBS for 15min with gentle inversion every 5min, followed by resuspension in a nuclei suspension buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2, 1% SUPERase In RNAse Inhibitor) and flash freezing in liquid nitrogen. For 10X sc-RNA-seq, cells were sorted for CD34+, CD43+ or CD45+ cells into 50% FBS using FACS (Aria III). For sci-RNA-seq, the samples were submitted to the Brotman Baty Institute for processing following a similar protocol to the one published in http://dx.doi.org/10.17504/protocols.io.9yih7ue. For 10X sc-RNA-seq, the manufacturer's protocol for library construction was followed.