title: GSM4200846: C11_2; Mus musculus; RNA-Seq
library_construction_protocol: 8 to 10-week-old C57BL6/J mice were subjected to myocardial infarction induced by permanent ligation of the left descendant coronary artery, or to sham surgery (i.e. mice underwent the exact same surgical procedure without coronary artery ligation). Cells were isolated from blood or ischemic heart of C57BL6/J mice in the following conditions: no surgery control (n=3), sham surgery day 1 (n=3), sham surgery day 3 (n=3), myocardial infarction day 1 (n=5), myocardial infarction day 3 (n=5). Venous blood was collected in EDTA containing collection tubes by retroorbital puncture using heparinized hematocrit capillaries and kept on ice with agitation until further processing. Red blood cells were removed by performing erythrocyte lysis in ACK buffer for 5' on ice twice. Ischemic cardiac tissue from mice with 1 and 3 days old infarcts was mechanically dissociated using a 12-Well Tissue Disaggregator plate (ScienceWare, Bel-Art Products).  Cells were labeled with hashtag antibodies 1 to 7 (TotalSeqA hashtag antibodies, Biolegend, at a final concentration of 2µg/ml), Fixable Viability Staining e780 (1:1000), B220-PE-Cy7 (clone RA3-6B2), NK1.1 PE-Cy7 (clone PK136), Ter119-PE-Cy7 (clone Ter119), CD11b-PercpCy5.5 (M1/70), Ly6G-PacificBlue (1A8), SiglecF-Biotin (clone EA798). At this step, CITE-seq antibodies against Ly6G (1.25µg/ml) and CD11b (1.25µg/ml) were also included in the labeling mix to avoid epitopes being inaccessible in the second CITE-seq labeling. Viable Ter119-B220-NK1.1-CD11b+ cells were sorted using a FACS Aria III (BD Biosciences) with a 100µm nozzle. The final content of Ly6G+ neutrophils and Ly6G- non-neutrophils in the sorted cells was adjusted to approximately 1:1 proportions. After sorting, cells were washed once and cells from each sample were pooled together (the volume of each sample used to pool cells was adjusted based on the number of sorted events, to ensure an approximately equal proportion of cells from each sample in the final single-cell RNA-seq data). Pooled cells were labeled with TotalSeq-A antibodies (Biolegend) directed against CD64 (5µg/ml), CD54/ICAM1 (5µg/ml), CD49d (5µg/ml), anti-biotin (2.5µg/ml), CD115 (2.5µg/ml), CD62L (1.67µg/ml), Ly6C (1µg/ml), IA-IE (1.25µg/ml), MSR1(5µg/ml), Fcer1a (5µg/ml), CCR3 (5µg/ml) and CXCR4 (5µg/ml), washed twice in PBS/0.04% BSA.  Hashtags/Sample list: Hashtag-1: blood cells, no operation control; Hashtag-2: blood cells, day 1 post sham surgery; Hashtag-3: blood cells, day 3 post sham surgery; hashtag-4: blood cells, day 1 post myocardial infarction surgery; hashtag-5: blood cells, day 3 post myocardial infarction surgery; hashtag-6: heart cells, day 1 post myocardial infarction surgery; hashtag-7: heart cells, day 1 post myocardial infarction surgery. Cells were loaded in the 10X Genomics Chromium at a concentration of 1700 cells/µl with the aim of recovering 15,000 cells. Libraries were generated with the Chromium Single Cell 3' Reagents Kit v3 Chemistry. Libraries were prepared with Chromium Single Cell 3' Reagents Kit v3 Chemistry and prepared for CITE-Seq/Hashing according to the manual until the cDNA amplification step in which 1 μl (0.2 μM) ADT PCR additive primer to capture Antibody-derived tags (ADTs) and 1 μl (0.1 μM) HTO PCR additive primer to capture Hashtag oligos (HTOs) were added. After cDNA amplification 60 μl (0.6x) SPRI beads (Beckman Coulter) were added to separate the supernatant fraction that contains the ADT/HTO-derived cDNAs (<180bp) and the bead fraction that contains the mRNA-derived cDNAs (>300bp). The mRNA-derived cDNAs (bead fraction) were processed following the standard 10x Genomics protocol. The ADT/HTO-derived cDNAs (supernatant fraction) were purified twice with 2x SPRI. After the two purifications half amount of the eluted cDNAs were amplified for ADTs and half for hashtags. In the same reactions, the ADTs were indexed using TruSeq Small RNA primers and the hashtags using modified TruSeq DNA primers. The libraries were purified once more with 1.6x SPRI (160 μl). The detailed protocol including the oligo sequences can be accessed here: https://cite-seq.com/protocol. Chromium single cell 3´v3 (10XGenomics)
attributes.tag: GEO Accession
attributes.value: GSM4200846