GSM5833244: wt3; Mus musculus; RNA-Seq
Crypt preparation: Approximately 5 cm of small intestinal tissue was dissected, longitudinally opened and extensively washed in PBS. Tissue was placed in 2 mM EDTA in PBS for 30 min on the rotator at 4C. Tissue fragments were placed in 10 mL of  0.1% BCS and pipetted up and down. The pipetting and 0.1% BCS solution exchange was repeated 6 times. Fractions 4-6 were collected and crypts were collected by centrifugation. Cells were flow-sorted as live single cells for single cell sequencing with 10X Genomicx. Single cells gene expression libraries were prepared by the McDonnell Genome Institute (MGI) at Washington University using the 3′ Single Cell v3 kit from 10x Genomics. Cells were partitioned into nanoliter Gel-bead-inEmulsions (GEMs) droplets. Each GEM underwent retro-transcriptase reaction to generate single-cell cDNA and received a unique 12-nt cell barcode and unique molecular identifier (UMI). GEM cDNA was amplified for 11 cycles before being purified using SPRIselect beads. For scRNAseq, 10 ml of purified cDNA was used to generate the library for RNA sequencing. Libraries were sequenced on a NovaSeq S4 (Illumina, 300 cycle kit) flow cell, targeting 50K read pairs per cell.