GSM7855410: Brain Organoid sc-RNA day 42 Rep1; Homo sapiens; RNA-Seq
Organoids were dissociated at day 18 or 42 by incubation in a 9:1 mixture of Accutase (Sigma Aldrich, cat # A6954) and 10x Trypsin (Gibco, cat # 15400) at 37 degrees on a Thermo-shaker (800rpm). Cells were filtered through a 35 um strainer followed by dilution with 0.1% BSA in DPBS-/- and stained with Draq7 viability dye (Biostatus; DR70250, 0.3 mM). Live, single, GFP positive cells were sorted with a BD FACSAria III (70 nm nozzle) and directly collected in a 96-well PCR plate pre-loaded with 10 uL of 0.5% BSA in DPBS-/-. A whole organoid was sorted to completion into a single well and the entire resulting cell suspension was subject to single cell transcriptomic analysis using the Chromium Single Cell 3′ Reagent Kit (v.3.1) following the 10X user guide with the following modification. During cDNA amplification (2.2), the PCR reaction is paused at 72 degrees after 6 cycles. 75 uL of sample is removed for targeted amplification (below) and replaced with 37.5 uL of Amp Mix, 11.25 uL cDNA Primers and 26.25 uL H2O before continuing for a total of 13 cycles