GSM9837477: PDO OKG146P, shControl and shZFP36L2 pooled together, IGFF, mRNA; Homo sapiens; RNA-Seq
Organoids were dissociated into single cells and incubated with TotalSeq hashtag antibodies (BioLegend, #B0251, #B0252, #B0253) for 30 minutes on ice. Viable (DAPI-negative) cells were sorted using a 130 μm nozzle on a SH800S cell sorter (Sony) into DPBS containing 0.04% BSA. Equal numbers of hashtag-labelled shZFP36L2 and shCtrl cells were pooled per condition and resuspended at 700–1,300 cells/μL for library preparation. Cell viability exceeded 90% as determined by Trypan Blue exclusion. Single-cell suspensions were loaded onto the Chromium Single Cell 3′ v3.1 platform (10x Genomics) according to the manufacturer's protocol, targeting up to 10,000 cells per sample for droplet-based encapsulation and barcoding. Following reverse transcription, emulsions were broken and cDNA was purified using Dynabeads MyOne SILANE, followed by PCR amplification. Libraries were sequenced on an Illumina NovaSeq platform with the following read configuration: Read 1, 28 cycles; i7 index, 8 cycles; Read 2, 90 cycles.