GSM5931737: hiPSCs cultured under 5iLAF conditions; Homo sapiens; RNA-Seq
Twenty-four hours before single-cell dissociation, 5iLAF human cells were treated with rock inhibitor Y27632 (5 µM - Axon Medchem #1683). Next day, human cells were dissociated using 1:1 TrypLE (Gibco #12605-028) 1X PBS and 0.25 mM EDTA (Invitrogen #AM9260G) for 5 min at 37 °C and resuspended in FACS buffer (1X PBS, 1 mM EDTA, 25 mM HEPES pH=7 and 1% BSA). Then, tdTomato+ human cells were FACS sorted with FACS AreaI and AreaII through a nozzle of 100 µm in 1X PBS 0.05% BSA, and the number of cells was quantified in a Neubauer chamber.  Cell suspensions were loaded onto a well of a 10X Chromium Single Cell instrument. Barcoding and cDNA synthesis were performed according to the manufacturer´s instructions. The cDNA libraries were constructed using the 10X Chromium Single cell 3' Library Kit v3 according to the manufacturer's original protocol and sequenced either with a NextSeq 500 (R1:28, R2:49, i7:8) instrument. RNA libraries were prepared for sequencing using standard Illumina protocols