Single nucleus RNA sequencing of an in vitro primary adult human colonic epithelium at early, mid, and late time points during long-term culture
Single nucleus RNA sequencing of an in vitro primary adult human colonic epithelium at early, mid, and late time points during long-term culture
Cells were gently scraped off the transwell membrane with a mini cell scraper in 200 µL of cold DPBS and transferred to a cryovial containing 1.3 mL of cold DPBS with a cut 0.1% BSA coated 200 µL pipette tip and centrifuged at 500 x g for 3 minutes at 4°C. Excess DPBS was gently removed from the cell pellet and the pellet was immediately flash frozen in liquid nitrogen. All samples were stored in liquid nitrogen until nuclei isolation. Single nuclei were isolated from flash frozen cell pellets using Chromium Nuclei Isolation kit (10x Genomics 1000493) and protocol for Single Cell Gene Expression and Chromium Fixed RNA Profiling (10x Genomics CG000505 Rev A) with added RNase inhibitor (2 U/mL, Roche 3335402001) throughout the isolation.  Because this protocol is designed for isolation of nuclei from whole tissue, we modified the initial lysis steps for gentler dissociation of the cell pellets by triturating the frozen cell pellet in cold lysis buffer 15 times with a 1 mL pipette and omitting the use of a pellet pestle. This was followed by a 5 minute incubation in the lysis buffer on wet ice with additional trituration at 2.5 minutes and 5 minutes of the incubation.  Isolated nuclei were counted and loaded into the 10x Chromium to create single-nuclei droplets targeting 5,000 nuclei. Single-cell libraries were prepared using the Next GEM Single Cell 3' Gel Bead kit v3.1 (10x genomics PN-1000147) according to the manufacturer's instructions by the University of Michigan Advanced Genomics Core.