title: GSM5415722: Sample 3_Resident cardiac macrophages_Day 28 AngII_Expt 1; Mus musculus; RNA-Seq
library_construction_protocol: (Samples 1-5, mouse): Hearts were perfused with 20 ml of cold PBS. Hearts were chopped finely and digested, while being shaken, at 37 °C in DMEM containing collagenase I (450 U ml–1), DNase I (60 U ml–1) and hyaluronidase (60 U ml–1) enzymes and 1mM Flavopiridol. Digestions were stopped after 30 min and cells were processed into a single-cell suspension on ice. Cells were labeled with CD45 magnetic beads (Miltenyi Biotec) and subsequently fluorescently tagged antibodies to CD45, CD64, CD11b, B220, CD3, Ly6G, NK1.1, Ly6C and MHC-II. Hematopoietic cells were positively enriched using the AutoMACS instrument (Miltenyi Biotec). Live resident macrophages (DAPI-CD45+CD64+CD11b+Td+) and monocytes (DAPI-CD11b+Ly6C+) were sorted on the Aria Fusion (BD Bioscience) under low pressure into DMEM containing 50% bovine serum for 10x Genomics single-cell RNA sequencing. Monocytes and macrophages were mixed in a 1:100 ratio and sequenced together. (Samples 6-7, human): Resected tissue was immediately placed into sterile, cold DMEM media supplemented with 50% fetal bovine serum. Human cardiac tissue was processed similar to mouse tissue and stained with anti-CD45 antibody. Cell sorting was performed using Aria II instrumentation (BD Bioscience). Total live immune cells were sorted by gating on DAPI-CD45+ cells. Samples were sorted directly into DMEM + 50% bovine serum for single-cell RNA sequencing submission. (Samples 1-5, 7): Libraries were prepared as outlined by the Chromium Single Cell 3' Reagent Kits v3 user guide with appropriate modifications to the PCR cycles based on the calculated cDNA concentration (as recommended by 10X Genomics). (Sample 6): Libraries were prepared as outlined by the Chromium Single Cell 3' Reagent Kits v2 user guide with appropriate modifications to the PCR cycles based on the calculated cDNA concentration (as recommended by 10X Genomics).