title: GSM8980198: CropSeq NTN 2, Sample 2, ADT; Mus musculus; OTHER
library_construction_protocol: Kidneys were incubated at 37 °C for 40 min in RPMI 1640 Medium (Thermo Fisher) containing 10% heat inactivated FBS, 1% P/S, 0.4 µg/ml Collagenase D (Roche, Basel, Switzerland) and 0.2 µg/ml DNAse (Roche). Subsequently, tissue was dissociated using the GentleMACS Dissociator and C-Tubes (both Miltenyi Biotec) and leukocytes were isolated using a gradient centrifugation with 37 % Percoll (GE Healthcare). After the centrifugation, cells were washed with DPBS (Gibco). CD4+ T cells were enriched using the EasySep Mouse CD4+ T cell Isolation Kit according to manufacturer's instructions (Stemcell Technologies, Vancouver, Canada). Enriched cells were sorted by Fluorescence-activated cell sorting (FACS) for CD4+ T cells, GFP signal and eBFP2 signal. For isolation of intestinal lymphocytes, the small intestine was obtained and cut into 1 cm pieces. We separated lamina propria lymphocytes (LPLs) from intraepithelial lymphocytes (IELs) by incubation with 2 mM DTT at 37°C for 30 min and subsequent filtering through a 100 µm cell strainer (Falcon). The IELs in the supernatant were discarded. The remaining parts were transferred into 50 ml tubes (Falcon, Thermo Fischer), containing DPBS, calcium, magnesium (Gibco), washed by rigorous shaking and repeatedly filtered through a 100 µm cell strainer. Further, the pieces were digested with collagenase D (Roche) and DNAse (Roche) at 37°C for 45 min while rotating in a MACS-Mix tube rotator (Miltenyi Biotec). Subsequently, the small intestines were minced in a MACS cell separator (C-tubes, program “intestine 01”) and filtered through a 70 µm cell strainer (Falcon) until the tissue turns white. The cell strainer was washed with RPMI-medium. Lastly, gradient centrifugation with 37% Percoll (GE Healthcare) was performed. Cells were sorted by Fluorescence-activated cell sorting (FACS) for CD4+ T cells, GFP signal and eBFP2 signal. All steps were performed according to standard protocol of the Chromium Single Cell 3' v3 kit, Chromium  Next GEM Single Cell 3' v3.1