title: scRNA-seq from Trex1 KO in tumor
design: scRNA-seq from Trex1 KO in tumor
library_construction_protocol: Tumors were weighed and homogenized using a tumor dissociation kit (Miltenyi Biotec, 130-096-730) with a GentleMACS (Miltenyi Biotec, program 37°C _m_TDK1). Following procedures previously described (J. Lim, R. Rodriguez, K. Williams, J. Silva, A. G. Gutierrez, P. Tyler, F. Baharom, T. Sun, E. Lin, S. Martin, B. D. Kayser, R. J. Johnston, I. Mellman, L. Delamarre, N. R. West, S. Müller, Y. Qu, K. Heger, The Exonuclease TREX1 Constitutes an Innate Immune Checkpoint Limiting cGAS/STING-Mediated Antitumor Immunity. Cancer Immunol. Res. 12, OF1–OF10 (2024).), for scRNA-seq, cells were incubated with Fc receptor block and labeled with TotalSeq-C hashtag antibodies (clone M1/42;30-F11, BioLegend, hashtag 1-6) together CD11b (clone M1/70, BD Biosciences, RRID:AB_312798), CD19 (clone with the following surface markers: 6D5, BioLegend, RRID:AB_313654), CD45 (clone 30-F11, BD Biosciences, RRID:AB_2870247), CD90.2 (clone 30-H12, BioLegend, RRID:AB_313176), NK1.1 (Clone PK136, BioLegend, RRID:AB_389364). Subsequently, cells were labeled with calcein blue (Invitrogen, C1429) and 7-AAD (BioLegend, 420403). Samples were sorted into T cells (CD45+CD90.2+CD11b-NK1.1-CD19-) and non-T immune cells (CD45+CD90.2-CD11b+NK1.1+CD19+) with a FACSAria instrument (BD Biosciences). The same number of T cells or non-T immune cells from each mouse per each population were pooled and loaded into a Chromium single cell sorting system (10X Genomics). Following procedures previously described (J. Lim, R. Rodriguez, K. Williams, J. Silva, A. G. Gutierrez, P. Tyler, F. Baharom, T. Sun, E. Lin, S. Martin, B. D. Kayser, R. J. Johnston, I. Mellman, L. Delamarre, N. R. West, S. Müller, Y. Qu, K. Heger, The Exonuclease TREX1 Constitutes an Innate Immune Checkpoint Limiting cGAS/STING-Mediated Antitumor Immunity. Cancer Immunol. Res. 12, OF1–OF10 (2024).), using the 10x Genomics Chromium Single Cell 5′ V2 Reagent Kit and Chromium Chip K Single Cell Kit, 4 samples of sorted single cell suspension were loaded onto the Chromium Controller. Cell counts and viability was checked using Cellometer K2 Image Cytometer cell count (Nexelom). Each sample was injected into microfluidic chips to form Gel Beads-in-Emulsion (GEMs) in the 10X Chromium X instrument. Reverse transcription (RT) was performed on the GEMs, and RT products were purified and amplified. DNA from hash tag oligos (HTO) was separated from cDNA based on size selection using SPRIselect beads (Beckman Coulter, B23318) and cDNA was used to generate Gene expression libraries. HTO libraries were generated using the 5' Feature Barcode Kit, (10X Genomics, PN-1000256). T cell receptor (TCR) libraries were amplified from the cDNA product (10X Genomics, PN 1000252) and enriched cDNA products were made into libraries using the library construction kit (10X Genomics, PN 1000190). Gene expression, TCR, and HTO libraries were then profiled using the Tapestation 4200 (Agilent Technologies) and quantified with Qubit Fluorometer (ThermoFisher Scientific).