GSM5600484: D13+12 SIX2Cre; Homo sapiens; RNA-Seq
Organoids were minced with a scalpel blade then dissociated in 1mL warmed Accutase, pipetting every 3 minutes for 15 minutes. The cell solution was placed on ice (2 minutes), passed through a 40um cell strainer, washed with TeSR-E6 and centrifuged (1500 rpm, 3 minutes). For D13 and D13+14 samples, barcoding was performed as described previously (Stoeckius M. et al., Genome Biol, 2018), incubating with 1µg hashtag oligo antibody (BioLegend TotalSeq-A0251 to A0258) for 20 minutes on ice prior to washing and pooling at equal cell ratios.  Prepared samples were resuspended in TeSR-E6 on ice and supplied for sequencing at a density of 300-1000 cells/uL. 10x libraries were constructed using 10x Chromium Next GEM Single Cell 3' Reagent kit v3.1. HTO librares were constructed following "BioLegend TotalSeq™-A Antibodies and Cell Hashing with 10x Single Cell 3' Reagent Kit v3 3.1 Protocol". Single cell RNA seq, with hastag oligo barcoding. Sequenced using Illumina Novoseq.