title: Illumina HiSeq 4000 sequencing; Single-cell RNA-seq of intestinal crypts of Red2Kras and Red2Pi3k mice
design: Single-cell RNA-seq of intestinal crypts of Red2Kras and Red2Pi3k mice
library_construction_protocol: Intestinal cell dissociation was performed as previously described with few modifications (Haber et al., 2017 and Stzepourginski et al., 2015). Briefly, proximal half of the small intestines were carefully washed with cold PBS and villi were scraped off using cover slip. Remaining tissues with crypts and lamina propria were then cut into 2 mm fragments and incubated in 10 mL Gentle Cell Dissociation Reagent (STEMCELL technologies) at 4 °C for 30 min. The fragments were then shaken vigorously and the supernatant was collected as first fraction in a new conical tube. The remaining fragments were further incubated in fresh Gentle Cell Dissociation Reagent and a new fraction was collected every 30 min. The first and second fraction were discarded because it may have some debris and villi. The third to fifth fraction contained the majority of the intestinal crypts. The crypts enriched fractions were then washed with 10 ml of cold PBS and filtered through a 100 µm cell strainer (Falcon). The crypts then further dissociated into single cells using TrypLE Express enzyme (GIBCO) at 37 °C for 5 min. The cells were filtered through a 40-μm cell strainer (Falcon). Remaining fragments with lamina propria were further digested with dissociation solution (2.5 mg/mL Liberase TL, Sigma; 10 U/mL DNAse, Promega) at 37 °C for 1 h. To help dissociation, we gently mixed tissue pieces by pipetting up and down every 10min. After 20 min, we harvested supernatants and added 1 volume of DMEM containing 10% bovine serum (BS, Sigma Aldrich) while adding 5 mL of fresh dissociation mix to the remaining tissue pieces. We repeated this step 3 times for a total time of 60 min. After completion of the 3 cycles, we mechanically disaggregated the remaining intestine fragments on a 100 μm mesh using a syringe plunger. The cell suspension was filtered through a 40 µm cell strainer into a 15 ml tube. The tube and filters were washed twice with 1 mL of 2% FBS in PBS and the cell suspension was then centrifuged at 300 g for 5 min. The supernatant was removed and the cell pellets were resuspended in 100 µL of antibody mix (2% FBS; 1:125 CD45 (30-F11)-APC; BD Biosciences; 1:125 EpCAM (G8.8)-PE-Cy7; BioLegend) and incubated for 1 h on ice. Both epithelial and lamina propria cells were then washed with 3 mL 2% FBS in PBS and filtered once more if clumps could be observed. After centrifugation at 300 g for 5 min the cell pellets were resuspended in 1 mL 2% FBS and 10 U/mL DNAse in PBS for sorting. MOFLO system (Beckman Coulter) was used for the sorting at Wellcome-MRC Stem Cell Institute Flow Cytometry Facility. Data were analysed with FlowJo software (Tree Star). All animals were injected with corn oil-diluted tamoxifen (dose 4mg/20g body weight) by intraperitoneal injection (IP) on the same date to achieve mosaic level of induction. Then they were culled at 2 weeks post induction and used for tissue collection.  Steps were performed according to manufacturers' instructions (10X Genomics, v2chemistry). Steps were performed according to manufacturers' instructions (10X Genomics, v2 chemistry).