Culture condition comparisons of WTC11 hiPSC by single-cell RNAseq and featuring differences between cE8, hE8 and B8+ media
Culture condition comparisons of WTC11 hiPSC by single-cell RNAseq and featuring differences between cE8, hE8 and B8+ media
Cells at 80% confluency were washed with PBS without calcium and magnesium and treated with TrypLE Express at RTroom temperature for 4 min. Following detachment, cells were washed in PBS supplemented with 1% bovine serum albumin (BSA) (Miltenyi Biotech, #130-091-376) and centrifuged at 100 × g for 5 min. Supernatants were removed, and cells were resuspended for antibody labeling according to the manufacturer's instructions (10x Genomics, User Guide CG000149 Rev D; protocol 2 for washing steps - page 9). Cells were incubated for 30 min on ice with TotalSeq-B hashing antibodies (BioLegend, TotalSeq -B0251, TotalSeq -B0252 ,TotalSeq -B0253, TotalSeq -B0254, TotalSeq -B0255, TotalSeq -B0256, cat. nos. #394631, 394633, 394635, 394637, 394639, 394641 respectively) diluted 1:50 in PBS + 1% BSA. Staining was performed in 2 ml low-bind Eppendorf tubes, in a final reaction volume of 100 µl for 1–2 × 1060^6 cells. Attached to this submission are the config_multi.csv and feature_ref.csv files, which contain the associations between sample names and hashtags, and hashtags and barcodes, respectively. After staining, cells were washed three times with PBS + 1% BSA and centrifuged at 100 g for 5 min between washes. Cells were then counted, and 4 × 105^5 cells per condition were pooled. The pooled suspension was centrifuged at 100 g for 5 min, resuspended in 1 ml PBS + 1% BSA, filtered through a 40 µm cell strainer, and recounted. 27600 multiplexed cells were loaded into the Chromium Controller (10x Genomics) for an expected recovery of 19000 final cells. Cells appeared live and round with >90% viability at the time of loading. 10X genomics technology with CG000732 RevA (with feature technology for surface protein). Cells were loaded on a chip G. The single-cell gene expression and ADT libraries were generated using the Chromium Single Cell 3ʹ v4 workflow (CG000732 RevA). GEM formation, reverse transcription (RT), and post-RT cleanup were performed by following the user guide. cDNA amplification, using Feature cDNA primers 2, was performed for 11 cycles. A Sample Index PCR with indexes TT (gene expression library) and NN (ADT library) was performed as suggested by the 10X guidelines. All libraries were purified using SPRIselect beads with 0.7x–0.9x double-sided size selection for the gene expression library or 1.2x single-sided size selection for the ADT library. All libraries (GEX and ADT) were quantified by Tapestation and Qubit dsDNA HS assays. 