title: GSM9463697: Heart CD45+ leukocytes, MI and NK depletion, ADT; Mus musculus; OTHER
library_construction_protocol: 5 minutes before being euthanasia, the mice were injected i.v. with 2.5µg anti-CD45.2 APC (clone 104, Biolegend, cat. #109814) under isoflurane anesthesia to label circulating immune cells. The heart was perfused with PBS, excised, and the infarct, peri-infarct area, and adjacent viable myocardium collected, weighed and digested for 1 hour at 37°C under agitation in RPMI containing 450U/ml collagenase I (Sigma-Aldrich, #C0130), 125U/ml Collagenase XI (Sigma-Aldrich, C7657), 60U/ml Hyaluronidase (Sigma-Aldrich, H3506), 60U/ml DNAse (Roche #11284932001). After digestion, the cell suspensions were passed through a 70µm cell strainer and washed twice in MACS buffer (PBS+0.5% BSA+2mM EDTA) and incubated for 5 minutes on ice with TruStain FcX™ (anti-mouse CD16/32, Biolegend #101320, 10µg/ml). Afterwards, cells were incubated with mouse CD45 microbeads (Miltenyi, 130-052-301, 1:10 final concentration), and anti-mouse CD45.2-AlexaFluor488. After 10 minutes incubation at 4°C, TotalSeq-A hashtag antibodies (Biolegend) were added as follows: hashtag 1 to 5 controls; hashtags 6 to 10: NK-depleted. Cells were incubated for an additional 15 minutes at 4°C, washed twice in MACS buffer, pooled, and magnetic selection was performed using LS Columns (Miltenyi (# 130-042-401) and MidiMACS separators (Miltenyi # 130-042-302). Sorted CD45+ cells were washed twice with PBS containing 0.04% BSA (Sigma-Aldrich A1595) and incubated for 25 minutes at 4°C in PBS/0.04% FCS containing Fixable Viability Dye eFluor™ 780 (ThermoFisher #65-0865-14, 1:1000) and anti-mouse TotalSeqA-Antibodies (ADT panel). Cells were washed with PBS/0.04% FCS, resuspended and viable CD45.2-AlexaFluor488+ CD45.2-APC- cells sorted using a BD FACS Aria III with a 100µm nozzle. Cells were resuspended, counted (final concentration 1,650 cells/µl). Genomics Chromium using Chromium Next GEM Single Cell 3' High Throughput (HT) Reagent Kits v3.1 were used for library construction with the aim to recover 40,000 cells.