GSM9428630: 80-day-old forebrain organoids, isogenic control; Homo sapiens; RNA-Seq
80-day-old forebrain organoids were washed with HBSS (STEMCELL Technologies) and incubated in dissociation solution (30 units/mL of activated papain (STEMCELL Technologies, prepared according to the manufacturer's instructions), 125 U/mL DNase I (New England Biolabs) in HBSS) at 37 °C on an orbital shaker at 90 rpm for 40 minutes with gentle mechanical dissociation using a micropipette every 10 minutes. Papain was then neutralized by the addition of 10 mg/mL ovomucoid protease inhibitor (Worthington) solution. Cells were washed with HBSS supplemented with 0.4% BSA (Proliant), passed through a 100 µm cell strainer (ThermoFisher) and resuspended at 1 x 10e6 cells/mL in HBSS 0.4% BSA. The concentration of the single-cell suspension as well as cell quality control were assessed with a Bio-Rad TC20 automated cell counter (Bio-Rad). An average of 3460 cells per sample was loaded onto the Chromium Controller (10x Genomics) to generate single-cell GEMs (gel beads in emulsion). GEM reverse transcription, Dynabeads cleanup (ThermoFisher Scientific), PCR amplification and SPRIselect beads cleanup (Beckman Coulter) were performed using the Chromium Single Cell 3' Reagent Kits v3.1 (10x Genomics) following the manufacturer's user guide. Indexed single-cell libraries were generated using the Chromium Next GEM Single Cell 3' Library and the Chromium Dual Index Kit. The quality of final indexed libraries was examined with a D1000 DNA Screentape on a TapeStation 2200 (Agilent Technologies) and the quantification was performed on the Qubit 3.0 fluorometer (ThermoFisher Scientific). The 10x Genomics libraries with unique indexes were pooled in equimolar ratios and sequenced for paired-end 100 bp reads on a NovaSeq 6000 (Illumina) at a depth of 82,500 read pairs per cell.