title: GSM8545814: Antibody Capture; Mus musculus; OTHER
library_construction_protocol: Spleens were mashed through 70-µm cell strainers with a syringe plunger and rinsed with PBS to prepare into single-cell suspensions. For kidney dissociation, we followed the protocol for mouse kidney dissociation using the Multi Tissue Dissociation Kit 2 from Miltenyi Biotec. To preserve cell surface epitopes, we decreased the volume of Enzyme P in the dissociation buffer and the kit's components for each kidney was as follows: 4.8 mL of Buffer X, 5 µL Enzyme P, 50 µL Buffer Y, 100 µL Enzyme D, and 20 µL Enzyme A. Kidney capsules were removed, and kidneys were placed into gentleMACS C tubes (Miltenyi Biotec) with dissociation buffer and quartered. GentleMACS Octo Dissociator with heating function (Miltenyi Biotec) was used and the program '37C_Multi_E' was run to dissociate kidneys. Cell suspension went through a 70-µm cell strainer and was rinsed with PBS. Red blood cells (RBCs) in spleen and kidneys were lysed with RBC lysis buffer (Invitrogen). Cells were resuspended in PBS and stained with LIVE/DEAD fixable near-IR dead cells stain kit (Invitrogen). Then cells were blocked with anti-mouse CD16/CD32 (BD Biosciences) followed by staining with the following antibodies: anti-CD3 epsilon-VioBlue (Clone 17A2, Miltenyi Biotec), anti-TCR β-Alexa Fluor 488 (Clone H57-597, BioLegend), anti-CD4-V500 (Clone RM4-5, BD Biosciences), and anti-CD8a-PE-Cyanine5 (Clone 53-6.7, BioLegend) in PBS with 1% fetal bovine serum (FBS). CD4+ T cells from a total of 10 Ezh2fl/fl mice and 9 iCD4-Cre Ezh2fl/fl mice were sorted by FACSAria cell sorter in the Unified Flow Core at the Department of Immunology in the University of Pittsburgh. CD4+ T cells isolated from a single organ in each mouse were individually incubated with unique TotalSeq-C anti-mouse antibodies from BioLegend. All CD4+ T cells were barcoded in the above manner to distinguish between different organs and mice before pooling cells together. Sequencing libraries of cell surface protein feature barcoding, single cell 5' gene expression, and paired single-cell T-cell receptor (TCR) were prepared according to instructions provided by10X Genomics in the Single Cell Core at the University of Pittsburgh.  These libraries were sequenced at the UPMC Genome Center. In total, 10,271 splenic CD4+ T cells from Ezh2fl/fl mice were sequenced, with a mean of 26,854 reads per cell, and 7,704 splenic CD4+ T cells from iCD4-Cre Ezh2fl/fl mice were sequenced with a mean of 22,603 reads per cell.  In the kidneys, 8,850 kidney-infiltrating CD4+ T cells from Ezh2fl/fl mice were sequenced, with a mean of 24,796 reads per cell, and 4,066 kidney-infiltrating CD4+ T cells from iCD4-Cre Ezh2fl/fl mice were sequenced with a mean of 25,649 reads per cell.