GSM9351035: hStrS-WTMg-8; Homo sapiens; RNA-Seq
For each condition, 4–5 organoids derived from three independent hiPSC lines were pooled. Organoids were dissociated into single-cell suspensions using an enzymatic solution containing papain (30 U/mL) and DNase I (0.4%) at 37°C for 45 min, followed by gentle trituration. Cells were washed in inhibitor-containing medium, filtered through a 70 μm strainer, counted, and resuspended in Cell Suspension Buffer for downstream single-cell RNA-seq library preparation. Single-cell RNA-seq libraries were generated using the PIPseq-T20 3' Single Cell RNA Kit v3.0 (Fluent BioSciences) according to the manufacturer's protocol. Briefly, ~40,000 cells per reaction were loaded into PIPseq Pre-templated Instant Partitions (PIPs) with partitioning reagents to encapsulate single cells with barcoded beads. Following cell lysis and mRNA capture in PIPs, cDNA was synthesized using a template-switch oligonucleotide, whole-transcriptome amplification was performed, and cDNA was purified by SPRI bead cleanup. Libraries were prepared by fragmentation, end repair, A-tailing, adapter ligation, and PCR amplification with dual indices, followed by size selection and QC on Qubit and Bioanalyzer/TapeStation. Libraries were sequenced on an Illumina NovaSeq S4 platform with 2×150 bp paired-end reads.