GSM5856681: scRNA data for mouse prostate rep1 dataset in Ext. Fig; Mus musculus; RNA-Seq
Tumor tissues were minced with a razor blade then digested in 280 U/mL Collagenase Type 3 (Worthington Biochemical) and 4 μg/mL DNase I (Sigma) in HBSS at 37°C for one hour and 15 minutes with periodic vortex every 20 minutes. Digested tissues were passed through 70 μm filters and pelleted. Cells were resuspended in 40% Percoll (Sigma) and layered above 60% Percoll. Sample was centrifuged at 1,900 g at 4°C for 30 minutes without brake. Cells at interface were collected, stained and analyzed by flow cytometry or sorting. Isolation of small intestinal intraepithelial lymphocytes has been previously described53. Briefly, small intestine between distal duodenum and proximal ileum was opened longitudinally and intestinal content was cleaned by washing in ice-cold PBS, followed by incubation in PBS/10 mM EDTA/1 mM Dithiothreitol solution at 37°C for 15 minutes with vigorous shaking. Tissues were passed through 100 m filters and pelleted. Cells were resuspended in 40% Percoll and centrifuged at 1,200 g at room temperature for 20 minutes. Cell pellets were collected, stained and analyzed by flow cytometry.  For flow cytometry experiments, cells were incubated with 2.4G2 mAb to block FcγR binding, DAPI (4, 6-diamidino-2-phenylindole; Sigma) or Aqua Live/Dead (Thermo Fisher Scientific) for the exclusion of dead cells and were stained with panels of antibodies for 30 minutes on ice. Granzyme B staining was carried out using the intracellular transcription factor buffer set from BD Pharmingen. All samples were acquired with an LSRII (BD) or LSR Fortessa (BD), and analyzed with FlowJo software version 9.6.2 (Tree Star). Cell sorting was performed with a FACSAria II (BD) using a 100 m nozzle. Tumor-infiltrating NK1.1+ abILTCks and PD-1+ T cells were sorted as CD45+TCRgd–TCRbeta+CD4–CD8alpha+PD-1–NK1.1+ and CD45+TCRgd–TCRbeta+CD4–CD8alpha+PD-1+NK1.1–, respectively. Thymic abILTCk/IEL progenitors and CD8 single positive cells were sorted as CD4–/dullCD8alpha–/dullCD1d/PBS-57–CD25–TCRbeta+CD122+CD5hiPD-1+NK1.1– and TCRbeta+CD4–CD8alpha+, respectively. For sorting of LSK cells, total bone marrow cells were incubated with CD117 MicroBeads (Miltenyi Biotec) according to the manufacturer's instruction, followed by positive selection with an LS column (Miltenyi Biotec) prior to staining with monoclonal antibodies. LSK cells were sorted as Lineage– (CD3epsilon–B220–Gr1–CD11b–Ter119–) CD117+Sca1+. RNA libraries were prepared for sequencing using standard Illumina protocols