library_construction_protocol: Single-cell preparation for single-cell RNA sequencing 1. Cardiac cells were isolated from mouse hearts 4 days after myocardial infarction using a neonatal heart dissociation kit Primary materials needed: Enzyme mixtures (Miltenyi Biotec, Cat# 130-098-373), surgery tools, butterfly (KRUUSE Butterfly, Cat# 121716), GentleMACS dissociator (Miltenyi Biotec, Cat# 130-093-235), ice-cold PBS, 10cm dish, 6-well plate, GentleMACS C-tube (Miltenyi Biotec, Cat# 130-093-237), 10cm injector, 70 μm strainer, 50ml tube, wide-bore tips. 1). Euthanize mice using CO2. 2). Perfuse the heart with 20 mL of ice-cold PBS. 3). Identify and cut the infarcted area, mainly the left ventricle. 4). Wash the tissue twice in ice-cold PBS, ensuring complete blood removal with tweezers. 5). Transfer the tissue to a 6-well plate with 2 ml of enzyme mixture. Chop the tissue into fine pieces using scissors and transfer each piece to a separate C-tube. Run the dissociator program once (gently shake if some tissue is stuck in the middle). 6). Follow the GentleMACS incubation protocol (15 min each, 3-4 times). 7). After incubation, spin down for a few seconds to let all the mixture settle at the C-tube bottom. Add 5 mL of medium to each C-tube and centrifuge at 1400rpm for 5 minutes at 4°C. 8). Remove the supernatant completely and suspend the pellet in 3.1 ml of ice-cold PBS for each C-tube, using 1 ml wide-bore tips. 9). Run the suspension through a 70 μm strainer, with a 15ml tube as a container. 2. Debris removal Primary materials needed: ice-cold PBS, Debris removal solution (Miltenyi Biotec, Cat# 130-109-398) 1). Add 900 μl of Debris Removal solution to each C tube. 2). Mix well by gently moving the pipette 10 times up and down using wide-bore tips. 3). Overlay very gently with 4 ml of ice-cold PBS. (Note: Tilt the tube and pipette very slowly to ensure the newly added PBS is not mixed with the cell suspension.) 4). Centrifuge at 1000g for 10 minutes at 4°C with full acceleration and full brake. Three phases will be formed. 5). Aspirate the two top phases thoroughly and gently using a vacuum and a 1 ml pipette. 6). Fill up with cold PBS to the final 15 ml. 7). Gently invert the tube three times. Do not vortex. 8). Centrifuge at 1000g for 10 minutes at 4°C. 9). Aspirate the supernatant completely. 3. Red Blood Cell Lysis Primary materials needed: MACS buffer (0.5% BSA, 2mM EDTA in PBS, pH 7.2) at room temperature (RT), RBC lysis buffer (Miltenyi Biotec, Cat# 130-094-183) at RT. 1). Resuspend the cell pellet in 1 ml MACS buffer at RT using wide-bore tips. 2). Add 10 mL of RBC lysis buffer at RT. Gently pipette up and down 10 times. 3). Incubate at room temperature for 2 minutes. 4). Centrifuge at 600g for 5 minutes at 4°C. Pipette off the supernatant completely. 4. Magnetic Labeling Primary materials needed: ice-cold MACS buffer, CD31 microbeads (Miltenyi Biotec, Cat# 130-097-418), CD45 microbeads (Miltenyi Biotec, Cat# 130-052-301), wide-bore tips (200um, 1ml), Maintain room temperature at 20 degrees Celsius (lower air conditioner). 1). Take one aliquot for calibration by FACS to determine the percentage of all cell types. 2). Mix microbeads before use. Incubate cell pellets with microbeads: 10ul CD45 + 10ul CD31 microbeads + 70ul MACS buffer. 3). Mix well and incubate microbeads for 15 minutes at 4-8 degrees (refrigerator, not on ice). Gently mix once every 5 minutes. Note: Working on ice may require increased incubation times. Higher temperatures or longer incubation times lead to non-specific cell labeling. 4). Add 1 mL of cold MACS buffer and centrifuge at 300g for 5 minutes at 4°C. 5). Remove the supernatant. Resuspend in 500 uL cold MACS buffer (Up to 108 total cells). Note: Work quickly, maintain cell coldness, and use pre-cooled solutions to prevent antibody capping on the cell surface and non-specific cell labeling. The volumes provided for magnetic labeling are for up to 107 total cells. For fewer than 107 cells, use the same volumes as indicated. Scale up all reagents and total volumes for higher cell numbers. 5. Magnetic Separation - This section is conducted in a cold room (4°C). Primary materials needed: MS column, OctoMACS separator (Miltenyi Biotec, Cat# 130-042-109), MACS buffer, Maintain a lower AC temperature (20°C). 1). Prepare the MS Column by rinsing with buffer: apply 500 μL of MACS buffer on top of the column and allow the buffer to run through. MS Columns are "flow stop," and should not run dry. (Filling with 500 μL takes ~1.5 minutes.) 2). Discard the effluent and change the collection tube. The MS Column is now ready for magnetic separation. 3). Apply the cell suspension onto the prepared MS Column. Collect the flow-through containing unlabeled cells. 4). Wash the MS column with 3X 500 μl of MACS buffer. Collect unlabeled cells that pass through and combine with the flow-through from the last step. (1st: ~1.5 min; 2nd: ~2 min; 3rd: ~3 min) Note: Perform washing steps by adding buffer aliquots as soon as the column reservoir is empty. 5). Remove the MS Column from the separator and place it on a new collection tube. 6). Pipette 1 mL of buffer onto the MS Column. Immediately flush out the fraction with the magnetically labeled cells by firmly applying the plunger supplied with the column. 7). Centrifuge the collected flow-through depletion cells (mainly cardiac fibroblasts)  and beads enriched cells (mostly immune cells) at 300g for 5 minutes at 4°C. 8). Resuspend the cells in 50-500 μl (adjusting based on the cell pellet size) ) of Cell Resuspension Buffer containing 0.04% BSA in PBS, pH 7.2. Count the cells and adjust the concentration to approximately 1X10⁶ cells/mL. 9). Combine 45 μl of depletion cells (~1X10⁶ cells/mL) with 15 μl of enriched cells (~1X10⁶ cells/mL) in one Eppendorf tube. Bring the samples for processing immediately. Note: Use the column immediately after filling to prevent the formation of air bubbles caused by warming up. Do not store columns after filling. The time required to fill the column with buffer depends on storage conditions, temperature, and humidity. Therefore, the filling time may vary from a few seconds to several minutes. This filling time does not influence the quality of the separation. Library preparation and sequencing of scRNA-seq scRNA-seq was performed using the Chromium Single Cell 3′ GEM, Library & Gel Bead Kit v3, 16 rxns (10x Genomics, PN-1000075) according to the manufacturer's instructions and targeting a recovery of 6,000 cells per dataset. Each dataset was obtained with a sample size of three mouse biological replicates. The libraries were constructed as instructed in the manufacturer's protocol. Sequencing was done on a Nova-Seq6000 using SP, 100 cycles kit mode allocating 800M reads in total (Illumina).The average read depth across the samples was 23,211 per cell.