Single-Nucleus Extraction from Tissue

Single-nucleus extraction from tissue was performed as previously described [55 (link)]. Briefly, engrafted colonic tissues were finely minced with a razor then transferred to a Dounce tissue homogenizer (Kimble Chase KT885300-0002) in 2 mL of ice-cold Nuclei EZ Lysis buffer (Sigma #N-3408) supplemented with protease inhibitor (Roche #5892791001) and RNase inhibitors (Promega #N2615, Thermo Fisher Scientific #AM2696). The tissue was ground 20–30 times with the loose pestle. The homogenate was filtered through a 200-μm cell strainer (pluriSelect #43-50200) then transferred back to the Dounce homogenizer, ground with the tight pestle 10–15 times. The homogenate was incubated on ice for 5 min with an additional 2 mL of lysis buffer, then filtered through a 40-μm cell strainer (pluriSelect #43-50040) and centrifuged at 500g for 5 min at 4 °C. The incubation and centrifugation steps were repeated one time, followed by resuspension Nuclei Suspension Buffer (1× PBS, 1% BSA, 0.1% RNase inhibitor) and filtering through a 5-μm cell strainer (pluriSelect #43-50005). The nuclei were then loaded onto the 10x Chromium Single Cell Platform for encapsulation and barcoding.