title: GSM7093909: Cardiac Macrophages Infarct 3; Mus musculus; RNA-Seq
library_construction_protocol: Mouse cardiac interstitial cells from healthy (control) or infarcted (7 days post-infarction) 12-16 week-old mice heart were obtained by enzymatic digestion. Briefly, heart tissue was minced and placed into a cocktail of Liberase TH (125 µg/mL) (Roche) and DNase (20 U/ml) (Sigma) in HBSS++ solution (Hanks balanced salt solution, Gibco) for 10 min at 37°C in an orbital shaker. After enzymatic incubation, tissue was mechanically digested by slowly pipetting to reach a single cell suspension. The supernatant was filtered through a cell strainer (40 μm, nylon; Falcon), the digestion was repeated once or twice with the remaining pieces, and the supernatants were pooled together. The total time for enzymatic digestion was around 30-45 min. Cell suspension was centrifuged for 20 min at 200g with centrifuge brakes deactivated to remove cell debris. Erythrocytes were removed using RBC lysis buffer (eBioscience). Cell suspension was then centrifuged at 500g for 5 min at 4°C, and resuspended in 100 µL of PBS containing 2% FBS and 2.5mM EDTA (sorting buffer). Cells in single cell suspensions were blocked with anti-mouse CD16/CD32 (1:250, BD Biosciences) for 15 minutes at 4°C. In order to identify neutrophils, the cell suspension was incubated for 30 minutes at 4°C in the dark with anti-Ly6G- PerCP/Cyanine5.5 (1:100, BioLegend). Cell suspension was washed twice with PBS/5% FBS, centrifuged at 500g for 5 min at 4°C, and the final pellet was resuspended in 200 µL of sorting buffer. Standard, strict forward scatter width versus area criteria were used to discriminate doublets and gate only singleton cells. Non-neutrophils (Ly6G-) were gated to identify EGFP+ macrophages, which were sorted with the FACSAria Sorter (BD Biosciences) in 1X PBS, 2% FBS. Single cell RNA-seq libraries were constructed using the Chromium System from 10X Genomics. Single Cell 3' Reagent Kit v3.1 (Single index) was employed for all the samples according to the manufacter's instructions. Briefly, the total of EGFP+/Ly6G- events were sorted in 1X PBS/2% FBS, and the number of cells was quantified in a Neubauer chamber. A maximum of 17,000 cells were loaded at a concentration of 1,000 cells/µL on a Chromium Controller instrument (10X Genomics) to generate single-cell gel bead-in-emulsions (GEMs). In this step, each cell was encapsulated with primers containing a fixed Illumina Read 1 sequence, followed by a cell-identifying 16 bp 10X barcode, a 12 bp Unique Molecular Identifier (UMI) and a poly-dT sequence. A subsequent reverse transcription yielded full-length, barcoded cDNA. This cDNA was then released from the GEMs, PCR-amplified and purified with magnetic beads (SPRIselect, Beckman Coulter). Enzymatic Fragmentation and Size Selection was used to optimize cDNA size prior to library construction. Illumina paired-end adaptor sequences (P5 beginning and P7 end) were added and the resulting library was amplified via end repair, A-tailing, adaptor ligation and PCR. Library quality control and quantification was performed using Qubit 2.0 Fluorometer (ThermoFisher Scientific) and Agilent's 4200 TapeStation System (Agilent), respectively. The libraries were also quantified by real time PCR (KAPA biosystems). Sequencing was performed in a Novaseq 6000 (Illumina) at the recommended configuration (Read1: 150bp; Index i7: 8bp each; Read2: 150bp) at an average depth of 50,000 reads/cell. Raw sequence data (.bcl files) generated from the sequencer were converted into fastq files and de-multiplexed using the 10X Genomics' cellranger mkfastq command.