GSM9455698: M3F_14v-1536 M3 Control; Mus musculus; RNA-Seq
The proximal third of the small intestine was harvested and gently irrigated in 5 ml of cold DPBS containing 1% BSA. The tissue was cut longitudinally and sliced into small fragments measuring 2 mm. These tissue fragments were incubated in 10 ml of ice-cold 20 mM RNase-free EDTA-DPBS for 60 min. During incubation, the tissue fragments were mixed every 15 min by pipetting through wide-bore tips. After the tissues fragments were incubated in EDTA-DPBS, the villus-rich supernatant was collected and filtered through a 30 µm MACS strainer. The filtrate was then centrifuged at 300 g for 5 min at 4°C in a swing bucket centrifuge, the supernatant was decanted, and the pellet washed twice in 10 ml DPBS containing 1% BSA. After the last wash, the pellet was set aside on ice. Concurrently, the tissue fragments were incubated in 5 ml TrypLE for 10 min at 37°C. After the TrypLE incubation, the tissue fragments were resuspended by pipetting through wide-bore tips; the crypt-rich supernatant was retrieved and filtered through a 30 µm MACS strainer. The sample was centrifuged at 300 g for 5 min at 4°C in a swinging bucket centrifuge, and the pellet washed twice in 10 ml DPBS containing 1% BSA. After the last wash, villus-rich and crypt-rich pellets were combined in 5 ml cold 0.04% BSA-PBS. Cell viability was immediately assessed using the Countess Automated Cell Counter; only samples with at least 80% viability and at least 1,000 cells/µl were used. Single-cell libraries were generated using the Chromium Single cell 3' GEM library from 10x Genomics