GSM4904123: HLO_Differentiation2_Replicate1; Homo sapiens; RNA-Seq
All tubes and pipet tips used for cell harvesting were pre-treated with 1% BSA in 1X PBS. The HLOs were transferred with a wide end pipet tip from matrigel to 1 mL Organoid Harvesting solution (Cultrex) and incubated 1h at +4C, with end-to-end rotation. Then the cells were dissociated in Accutase (Stem Cell) with DNaseI (200 U/mL, Worthington) and Dispase (100 U/mL, Corning) at 37C, in 10 min increments, with end-to-end rotation, until single cell suspension was obtained. The cells were washed in Cell Staining Buffer (Biolegend) and stained with TotalSeq HTO antibodies for 30 min on ice. The cells were washed twice in Cell Staining Buffer and filtered through a 40 µm pipet tip strainer (BelArt). The cells were counted using Trypan Blue dye and hemocytometer and pooled for sequencing. scRNA-seq libraries were prepared with Chromium Next GEM Single Cell 3′ Kit v3.1 (10x Genomics), with custom amplification of TotalSeq HTO sequences (Biolegend). The libraries were sequenced on Illumina NovaSeq sequencer in the Center for Advanced Technologies (UCSF).