title: GSM9232293: Human TS cell, stem state,scRNA-Seq, replicate 1; Homo sapiens; RNA-Seq
library_construction_protocol: TS cells were collected in the stem state (D0) and at days 3, 6, and 8 (D3, D6, D8) of EVT cell differentiation. At the time of harvest, cells were incubated with TrypLE (12604039, Gibco) for 15 min at 37°C. Detached cells were then neutralized with 0.3% BSA in PBS, centrifuged for 3 min at 500 × g, and resuspended in 0.3% BSA in PBS. Cells were washed three times with 0.1% BSA in PBS. Cells were counted, and viability was assessed prior to submission for library preparation. The 10X Genomics Single Cell 3' Expression v3.1 library preparation was performed using the 10X Genomics Chromium X. The second strand cDNA amplification was performed using the cDNA Amplification Mix on the Eppendorf MasterCycler Pro. The QC and quantification of the cDNA was determined using the Agilent TapeStation 4200 HS D5000 ScreenTape assay (5067-5592, Agilent). Validation of the single cell library was conducted using the Agilent TapeStation 4200 D1000 ScreenTape assay. Single cell library quantification was completed using a Roche LightCycle 96 using FastStart Essential Green Master (06402712001, Roche) and KAPA Library Quantification DNA Standards (KK4903, Illumina). Library concentrations were adjusted to 2 nM and pooled. The library pool was diluted for final on-board clustering concentration of 130 pM on a NovaSeq 6000. The sequencing was performed using a NovaSeq 6000 100 cycle Reagent Kit v1.5 (20028401, Illumina) and NovaSeq X Plus 100 cycle Reagent Kit (20104703, Illumina) with an asymmetrical sequencing profile (read 1 - 28 cycles: i7 index read - 10 cycles: i5 index read - 10 cycles: read 2-90 cycles).