Single-cell RNA-sequencing of wild-type, I830077J02Rik, C130074G19Rik and A530016L24Rik gene knockouts at day 7 of embryoid body differentiation
Single-cell RNA-sequencing of wild-type, I830077J02Rik, C130074G19Rik and A530016L24Rik gene knockouts at day 7 of embryoid body differentiation
Embryoid bodies at day 7 (D7) of differentiation were washed three times with PBS by spinning at 1000 rpm for 5 minutes, and dissociated using TrypLE (Gibco, 12604013) in a 37°C water bath while shaking with intermitting pipetting to facilitate dissociation. After 5 minutes, Trypsin/EDTA was added and dissociation continued for another 1 minute while pipetting. The dissociation was inactivated with PBS supplemented with 10% FBS (PBS-FBS), strained with a 40 µM cell strainer, washed three times with PBS-FBS by centrifuging at 300 rpm for 10 minutes and prepared for downstream applications.  Viable single cells (18000 per run, with an estimated recovery of 10000 cells) were encapsulated for cDNA synthesis and barcoded on a Chromium X using the Chromium Single Cell G Chip and final libraries were generated using the 10X Chromium Next GEM Single Cell 3' kit v3.1 (10X Genomics) and instructions.  Final libraries were generated using the 10X Chromium Next GEM Single Cell 3' kit v3.1 (10X Genomics) and instructions. Quality and quantity of the sequence libraries were determined with a High Sensitivity DNA kit using an Agilent 2100 Bioanalyzer (Agilent). 