title: GSM5917431: F1_2 [HTO]; Mus musculus; RNA-Seq
library_construction_protocol: Mice were treated daily with a monoclonal anti-CCR2 (MC-21, i.p. 20µg/mouse/injection) or with an isotype control antibody (rat IgG2b, clone LTF-2, BioXcell # BE0090) starting 1 day before the induction of myocardial infarction (MI) to deplete circulating CCR2+ monocytes. Mi was induced by permanent coronary artery ligation. Control mice (steady state heart, n=3), day 5 post-MI isotype control treated mice (n=4), and day 5 post-MI anti-CCR2 treated mice (n=5) received an i.v. injection of 2.5µg anti-CD45.2 APC (clone 104, Biolegend, cat. # 109814) under isoflurane anesthesia. The heart was perfused via intracardiac injection of PBS, excised, the right ventricle removed, and the infarct, peri-infarct area, and adjacent viable myocardium were 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). Cells were 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 anti-VSIG4-PE (clone NLA-14, final concentration 2µg/ml, ThermoFisher #12-5752-82),  anti-LYVE1-Biotin (clone ALY7, 5µg/ml, ThermoFisher #13-0443-82), mouse CD45 microbeads (Miltenyi, 130-052-301, 1:10 final concentration), anti-mouse CD45.2-eFluor506 (steady state heart samples, clone 104, 2µg/ml, ThermoFisher #69-0454-82), anti-mouse CD45.2-AlexaFluor488 (MI Day 5 isotype samples, clone 104, 2µg/ml, Biolegend #109816), anti-mouse CD45.2-BV421 (MI Day 5 isotype samples, clone 104, 2µg/ml, Biolegend #109832). After 10' incubation at 4°C, TotalSeq-A hashtag antibodies (Biolegend, see table for CITE-seq panels) were added as follows: Hashtag 1 to 3: steady state heart; Hashag 4 to 7: MI Day 5, isotype control treated; Hashtag 8 to 12: MI Day 5, anti-CCR2 treated. 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' 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. Cells were washed with PBS/0.04% FCS, resuspended and sorted using a BD FACS Aria III with a 100µm nozzle. Number of sorted cells was adjusted so that cells from steady state hearts (CD45.2-e506+), MI Day 5 isotype-treated hearts, and MI day 5 anti-CCR2-treated hearts represent 20%, 40% and 40% of all cells, respectively. Cells were resuspended, counted (final concentration 1,200 cells/µl) and 20,000 cells were loaded in the 10x Genomics Chromium in duplicate lanes, and scRNA-seq ADT and HTO libraries were prepared. Single cells were encapsulated into droplets with the Chromium™ Controller (10x Genomics). Transcripts captured in all cells encapsulated with a bead were uniquely barcoded using a combination of a 16 bp 10x Barcode and a 12 bp unique molecular identifier (UMI). cDNA libraries were generated using the Chromium™ Single Cell 3' Library & Gel Bead Kit v3 (10x Genomics) following the detailed protocol provided by the manufacturer. Chromium Single Cell 3' v3 (10X Genomics)