GSM7029391: Sox9 HET; Mus musculus; RNA-Seq
Isolated intestines from euthanized mice were washed with ice-cold PBS; villi were scraped using glass slides and dissociated in 5mM EDTA in PBS at 4ºC for 20 minutes, shaking every 5-7 minutes for about 30 seconds. The epithelial fraction was collected by centrifugation and incubated at 37ºC for 30 minutes in pre-warmed 4x TrypLE. Single cells filtered through 70um filter post trypsinization, washed with complete media, and collected in FACS buffer. About 17,000 viable cells were loaded onto a 10X Genomics ChromiumTM instrument (10× Genomics) according to the manufacturer's recommendations.  The scRNAseq libraries were processed using ChromiumTM single cell 5' or 3' library & gel bead kit (10× Genomics). Matched cell hashing libraries were prepared using single cell 5' feature barcode library kit. Quality controls for amplified cDNA libraries, cell hashing libraries, and final sequencing libraries were performed using Bioanalyzer High Sensitivity DNA Kit (Agilent). The sequencing libraries for scRNAseq and scTCRseq were normalized to 4nM concentration and pooled using a volume ratio of 4:1. The pooled sequencing libraries were sequenced on Illumina NovaSeq S4 300 cycle platform. The sequencing parameters were: Read 1 of 150bp, Read 2 of 150bp and Index 1 of 8bp. The sequencing data were demultiplexed and aligned to mm10 or hg19-3.0.0 using cell ranger version 3.1.0 pipeline (10× Genomics). 