GSM5718074: 3307_wt_CDHFD; Mus musculus; RNA-Seq
Small intestines were harvested and cleaned of fat residue and Peyer's patches. To obtain lamina propria cells (LPC), the small intestine was cut longitudinally and scraped in PBS 1X to clean the mucus and feces. The clean tissue was fragmented into pieces in 20ml HBSS 2% FCS. The pieces were filtered through a 100μm mesh and were incubated in 10ml HBSS 2mM EDTA in a shaker at 37°C for 20 min. The pieces were rinsed with HBSS through a 100μm mesh and incubated in 10ml HBSS 2mM EDTA in a shaker at 37°C for 20 min. The pieces were rinsed with HBSS, filtered through a 100μm mesh and incubated in 15 ml IMDM +10% FCS containing 1mg/ml collagenase VIII (Sigma) in a shaker at 37°C for 15 min in order to disaggregate the tissue. Single cell suspensions were then washed and filtered filtered (100 and 40μm cell strainer). Staining was performed using Live/Dead discrimination by using DAPI or ZombieDyeNIR according to the manufacturer´s instructions. After washing (~400g, 5min, 4°C), cells were stained with anti-CD45 antibody (Alexa Fluor® 700 anti-mouse CD45 Antibody, cat.no.103128, concentration 1:200) for 20min at 4°C and washed again. Samples for flow cytometry were fixed using eBioscience IC fixation (cat.no.00-8222-49) according to the manufacturer´s instruction. For sorting, a FACS Aria II and a FACSAria FUSION were used. After the isolation the cells were washed once with cold PBS + 0.1% KnockOut™ Serum Replacement (cat.no.10828010, TermoFisher Scientific) and subjected to single-cell RNA sequencing analysis using the Chromium System (10x Genomics, California, USA). 10x genomics according to the manufacturer protocol single cell RNA-seq 3'