scRNAseq from iPSC lines derived from a healthy donor and a DMD patient differentiated into the myogenic lineage
scRNAseq from iPSC lines derived from a healthy donor and a DMD patient differentiated into the myogenic lineage
Cells were filtered at 70µM and washed (500g, 5min, 4°C) in PBS then counted and checked for viability. They were resuspended in 100 uL of staining buffer (1xPBS with 2%BSA, 0,01% Tween20, 0,22µM filtered) and incubated with 10 µL human FcR blocking reagent (Miltenyi Biotec 130-059-901) for 10 min on ice. 1 µL of anti-human Totalseq-B Hashtag oligonucleotides antibodies (HTOs) (Biolegend, San Diego, USA) were assigned to each sample and incubated for 30 min on ice. Tagged cells were then washed three times in 1mL of staining buffer (500g, 5min, 4°C). Cells were counted and checked for viability, then pooled in equal proportions per sample. The viability of the pools varied between 71-98%.  A total of 20000 cells per pool were loaded on the Chromium Next Controller using the Chromium Next GEM Single Cell 3ʹ GEM, Library & Gel Bead Kit v3.1 (PN-1000121, 10x genomics, Pleasanton, USA). We followed the supplier's user guide CG000206 Rev D and generated 3'GEX and Cell Surface (CS) libraries using 3' feature barcode kit (PN-1000079) during pre-amplification step. For PCR index, we used Single Index Kit T Set A, 96 rxns PN-1000213 for 3'GEX CS. All libraries were checked on a Bioanalyzer with a High Sensitivity chip (Caliper Perkin Elmer, Waltham, USA). As expected, 3' GEX libraries peaked around 350pb and CS at 220pb