title: CT26 sorted in vivo tumor cells with Trex1 KO
design: CT26 sorted in vivo tumor cells with Trex1 KO
library_construction_protocol: Tumors were collected 10 days after tumor inoculation. Tumors were weighed and homogenized using a tumor dissociation kit (Miltenyi, 130-096-730) with a GentleMACS (Miltenyi, program TDK1). Cell suspensions were incubated in Fc receptor block (BD Biosciences, 2.4G2, #553142, RRID:AB_394657) and fixable viability dye (Invitrogen, 65-0865-14) in PBS. Cells were labeled with surface antigens for 20-30min at 4째C. For transcription factor staining, cells were fixed with FoxP3 Fixation Buffer (005523, eBioscience) for 30 minutes in the dark at room temperature. For cytoplasmic protein labeling, cells were fixed with Fixation buffer (554714, BD Biosciences) for 20 min at 4째C. Then, cells were stained with intracellular antibodies for 30min at 4째C. The following antibodies were used: BST2 (clone 927, BioLegend, RRID:AB_2562477), CD4 (clone RM4-5, BD Biosciences, RRID:AB_2870166), CD8 (clone KT15, MBL, RRID:AB_10597265), CD11b (clone M1/70, BD Biosciences, RRID:AB_2874105), CD11c (clone HL3, BD Biosciences, RRID:AB_2738580), CD19 (clone 6D5, BioLegend, RRID:AB_11218994), CD44 (clone IM7, BD Biosciences, RRID:AB_2870671), CD45 (clone 30-F11, BD Biosciences, RRID:AB_2870247), CD49b (clone DX5, BD Biosciences, RRID:AB_11153857), CD90.2 (clone 30-H12, BioLegend, RRID:AB_493725), F4/80 (clone BM8, BioLegend, RRID:AB_2564588), GZMB (clone GB12, Invitrogen, RRID:AB_10373420), LY6C (clone HK1.4, BioLegend, RRID:AB_2562351), LY6G (clone 1A8, BioLegend, RRID:AB_2566317), MHC-ll (clone M5/114.15.2, BD Biosciences, RRID:AB_2738192), and SCA-1 (clone D7, BD Biosciences, RRID:AB_2871229). Data were acquired on a Symphony flow cytometer (BD Biosciences) or Cytek Aurora (Cytek Bioscience) and analyzed using FlowJo software (FlowJo LLC, RRID:SCR_008520).   For scRNA-seq, cells were incubated with Fc receptor block and labeled with TotalSeq-C hashtag antibodies (clone M1/42;30-F11, BioLegend, hashtag 1-12) together with the following surface markers: CD45 (clone 30-F11, BD Biosciences, RRID:AB_2870247), CD90.2 (clone 30-H12, BioLegend, RRID:AB_493725), CD31 (clone MEC13.3, BioLegend, RRID:AB_2572181), and PDGFRa (APA5, BioLegend, RRID:AB_2043969). Subsequently, cells were labeled with calcein blue (Invitrogen) and 7-AAD (BioLegend).  Samples were sorted for tumor cells (CD45-Pdgfra-CD31-) with a FACSAria instrument (BD Biosciences).   Snap frozen mouse tissues were homogenized with nuclease free hard tissue homogenizing mix 2.8 mm ceramic beads (OMNI International) by using a Bead Mill Homogenizer (OMNI International) in RLT buffer. Tissue homogenates were centrifuged at 13,000 rpm for 15 min at 4째C and the supernatants were proceeded for RNA isolation with RNeasy Plus Mini kit (Qiagen). Total RNA was quantified with the Qubit RNA HS Assay Kit (Thermo Fisher Scientific) and quality was assessed using RNA ScreenTape on a TapeStation 4200 (Agilent Technologies). For sequencing library generation, the TruseqStranded mRNA kit (Illumina) was used with an input of 100-1000 nanograms of total RNA. Libraries were quantified with Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and the average library size was determined using D1000 ScreenTape on TapeStation 4200 (Agilent Technologies).