GSM5931393: HTO_G8_2; Mus musculus; RNA-Seq
Myocardial infarction (MI) was induced by permanent coronary artery ligation in Trem2+/+ (wildtype) and Trem2-/- mice. Non-infarcted wildtype mice (n=2), non-infarcted Trem2-/- mice (n=3), wildtype mice at day 5 after myocardial infarction (n=5) and Trem2-/- mice at day 5 after myocardial infarction (n=5received 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-AlexaFluor488 (Trem2+/+ heart samples, clone 104, 2µg/ml, Biolegend) or anti-mouse CD45.2-Alexa700 (Trem2-/- heart samples, clone 104, 2µg/ml, Biolegend). After 10' incubation at 4°C, TotalSeq-A hashtag antibodies were added as follows: Hashtag 1-2: WT no MI; Hashtag 3, 4 and 15: Trem2-/- no MI;  Hahstag 5, 6, 7, 8, 9: wildtype MI day 5; Hashtag 10, 11, 12, 13, 14: Trem2-/- MI day 5. 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. Cells from steady state hearts (~50% Trem2+/+ and ~50% Trem2-/-) and from day 5 post-MI hearts (~50% Trem2+/+ and ~50% Trem2-/-) were sorted separately and pooled post-sort so that steady state and day 5 post-MI cells represent ~30% and ~70% of the total cell pool, respectively. 20,000-25,000 cells were loaded in the 10x Genomics Chromium in duplicate lanes. 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.