title: GSM8526961: Axin2+EpCAM+ cells from adult small intestinal crypts of Axin2-CRE-ERT_Rosa-tdTomato mice; Mus musculus; RNA-Seq
library_construction_protocol: The mouse small intestines were dissected in 3 equal pieces. The small intestines were cut in pieces of 4 mm2, placed into 50 ml conical tubes and washed 3 times for 20 min with 25 ml of PBS/ 5 mM EDTA on a rocking plate at room temperature. Villi were mechanically removed by gentle shaking 3 times and trituration using a 25 ml pipette. Crypts were separated from the intestine by vigorous shaking 2 times. Crypts from all three parts were collected by centrifugation at 200g for 5 min, washed twice with PBS. Crypts from three Axin2Cre-ERT2:Rosa26tdTomato mice were mixed at equal ratios. To isolate epithelial cells, single-cell suspensions were stained with APC–conjugated anti-EpCAM 1:1000 (eBioscience) antibody for 30 min at room temperature. Fluorescence-activated cell sorting was performed using BD FACS Aria III SORP cell sorter (100 μm nozzle). Living cells were gated by DAPI dye exclusion. Fifty thousand single EpCAM+ cells were sorted in PBS supplemented with 0,4% BSA. Capturing of single cells was performed using the BD Rhapsody system (BD Biosciences). 15000 cells were loaded on a BD Rhapsody cartridge. Capture of single RNA molecules on barcoded magnetic polydT- beads and cDNA synthesis was performed as described in the manual (Single cell capture and cDNA synthesis with the BD Rhapsody single-cell analysis system; Doc ID: 210966 Rev. 1.0). BD's WTA kit was used for library preparation according to the manual. cDNA libraries were prepared following the manual (mRNA Whole Transcriptome Analysis (WTA) and sample tag library preparation protocol). Upon purifying cDNA libraries, concentration of libraries was assessed by a Qubit flex system using the DNA high sensitivity kit and average library size was determined on a bioanalyzer using a high-sensitivity DNA chip. Final sequencing library was prepared by dilution and pooling cDNA following the recommendations of the sequencing preparation sheet provided by BD. The libraries were sequenced on a Novaseq6000 (Illumina).