Single cell transcriptomics of organoids
Organoids were pooled and each cell aggregate were minced using a sterile scalpel. The dissociated cells were strained using 40 µm cell strainer and collected in a 15 ml falcon tube. The cells were kept on ice and final concentration was approximately 1000 cells/µl with > 90% cell viability. scRNA-Seq cDNA library was prepared using the chromium single cell system (10X Genomics). 10,000-16,000 cells per sample were added to a single cell master mix, following the Chromium Single Cell 3’ Reagent kits v3.1 protocol. The libraries were sequenced paired end on Illumina NovaSeq 6000 to a read depth of more than 30,000 reads per cell. All samples had sequencing yields of more than 143 million read per sample. The sequencing run was setup as a 28 cycles + 90 cycles non-symmetric run. Demultiplexing was done allowing 1 mismatch in the barcodes. Over 95.5% of bases in the barcode regions have Q30 or above and at least 93.2% of bases in the read have Q30 or above. More than 95.4% of bases in the UMI have Q30 or above. The analysis was performed with the Cell Ranger 6.1.2 software using the default parameters. Cells with extremely low number of UMI counts were filtered out. The aggregated.tar files contain the gene matrix files for performing single cell-RNAseq analysis.
Steps to reproduce
Codes for scRNA seq analysis are available in the github file: https://github.com/sarthak-sahoo-0710/Organoid_scRNASeq_analysis.