GSM6742619: Wild Type, biological replicate 2, scRNAseq; Mus musculus; RNA-Seq
Organoids were individually seeded in a 384-well plate and cultured for 7 days before processing. On day 7, the organoids were retrieved from the matrigel using ice-cold PBS, centrifuged at 300 g for 5min and incubated in TrypLE for 15min. The dissociated organoids were then passed through a 35 µm cell strainer and the flow through centrifuged at 300 g for 5min. The pellet was resuspended in 1mL Cell Recovery Solution (Sigma Aldrich, DLW354253) and incubated for 2h at 4°C under constant rotation. Samples were then centrifuged at 300 g for 5min and the pellet resuspended in 1 mL 0.04% BSA (in PBS). The pellet was resuspended in 100 µl 0.04% BSA and dissociated organoids were passed through a 35 µm cell strainer. Filtered cell suspensions were then counted using an automated cell counter, diluted to a concentration of 1 million total cells / mL in 0.04% BSA and kept on ice until encapsulation. scRNA-seq was conducted using a 5' scRNA-seq gene expression workflow (Chromium Single Cell Immune Profiling, Solution v1.1, 10x Genomics). Following encapsulation of cells using the Chromium Controller, GEM-RT, cDNA amplification, and construction of final libraries was conducted following manufacturer's instructions. Size profile and concentration of final libraries were assessed by the Agilent 2100 Bioanalyzer (High Sensitivity DNA Kit, Agilent, 5067-4626) and Qubit (BR DNA Assay, ThermoFisher, Q32853), respectively. Sequencing of final libraries was conducted on the Illumina NextSeq 550 10x Genomics recommendations (26 cycles read 1, 8 cycles i7 index, 98 cycles read 2, targeting a minimum depth of 20,000 reads/cell).