GSM9019670: Human BE organoid line WU014 Hindgut group; Homo sapiens; RNA-Seq
For human Barrett's esophagus organoid single-cell expression profiling, organoids were pelleted and digested to single cells using TrypLE (Gibco) digestion and manual pipetting dissociation, and then neutralized with Advanced DMEM/F12 (Gibco), 10% FBS (Gibco), 1X HEPES, 1X L-glutamine, 1X Pen/strep, 10 µM Y-27632 (Biogems), and 10 µM Primocin (InvivoGen). Cells were filtered using a 30 µm filter, counted, and volume adjusted before submission to the Washington University Genome Technology Access Core (GTAC). The single-cell gene expression Library was prepared according to the Chromium Single Cell Gene Expression 3'v3 instruction (10x Genomics). Briefly, single cells, reverse transcription (RT) reagents, Gel Beads containing barcoded oligonucleotides, and oil were loaded on a Chromium controller (10x Genomics) to generate single-cell GEMS (Gel Beads-In-Emulsions) where full-length cDNA was synthesized and barcoded for each single cell. Subsequently, the GEMS are broken and cDNA from each single cell is pooled. Following cleanup using Dynabeads MyOne Silane Beads, cDNA is amplified by PCR.  The amplified product is fragmented to optimal size before end-repair, A-tailing, and adaptor ligation. The final library was generated by amplification. After passed the quality control, the next-generation sequencing of libraries was performed on NovaSeq 6000 (Illumina).