GSM7990650: Transplanted_mouse_lung, hashtagged, genome sequencing2; Mus musculus; OTHER
Transplanted left lungs and recipient spleens from four mice were collected 7 days following transplantation. Two samples were collected at a time and pooled for further processing. Lung tissue was digested, and single-cell suspensions were prepared as previously described (PMID: 28615357). Cells were stained using DAPI (BD Biosciences, #564907) and were sorted by flow cytometry for CD45+CD4+Foxp3+ Tregs into 250 μL cell resuspension buffer (0.04% BSA in PBS). Collected cells were centrifuged (300 rcf for 5 min at 4 °C) and resuspended in collection buffer to a target concentration of 1,000 cells/μL. Cells were counted on a hemocytometer before proceeding. Single-cell suspensions of sorted lung and spleen Tregs were submitted to the Genome Technology Access Center core facility (Washington University in St. Louis) for single-cell GEM construction and cDNA synthesis and library construction. Two sets of hashtagged samples were submitted as follows: tolerant lung CD4+, tolerant lung Treg, tolerant spleen Treg, rejecting lung Treg. Sorted cell types were labeled using a TotalSeq anti-mouse hashtag antibody (Biolegend, #155861, #155863, #155865, #155807) prior to pooling for sample submission. Samples were processed using the Chromium Single Cell 3′ Library & Gel Bead Kit (10X Genomics, v3) and Chromium Single Cell V(D)J Reagent Kits (10x Genomics) following manufacturer's protocols. The libraries were sequenced on NovaSeq S4 (Illumina) targeting 50,000 reads per cell and 500 million read pairs per library. Cells were aligned to the mouse mm10-2020-A transcriptome using CellRanger (10× Genomics, v6.1.1) to generate feature-barcoded count matrices.