GSM8595765: Cryoablation nonmyocytes Day28 scRNA-seq GEX; Mus musculus; RNA-Seq
Surgery / Cryo / LAD / Sham LV cryoinjury (Cryo), ischemia-reperfusion injury after left anterior descending artery (LAD) ligation, and sham surgery were applied to female wildtype C57BL/6J mice at 12 weeks of age. Surgery and cryoablation were performed as described previously (Simon-Chica et al. 2024). In brief, mice were anaesthetized by intraperitoneal injection of 80–100µL of anesthesia solution (20mg/mL ketamine (Ketaset; Zoetis, Parsippany-Troy Hills, NJ, USA), 1.4mg/mL xylazine hydrochloride (0.12% Rompun; Bayer, Leverkusen, Germany), in 114 mM NaCl (0.67% m/v; B. Braun Melsungen, Melsungen, Germany)). After induction of deep anesthesia, eye ointment (Bepanthen containing 50mg/mL dexpanthenol; Bayer) was applied, 500µL glucose solution (278mM glucose = 5% (m/v); B. Braun Melsungen) was injected intraperitoneally, and 250µL of analgesia solution (10µg/mL buprenorphine (Temgesic; Indivior Inc., North Chesterfield, VA, USA) in 154 mM NaCl (0.9% m/v, B. Braun Melsungen)) was injected subcutaneously in the neck. Mice were shaved at the left side of the thorax (precordial region) and on the right leg. Mice were placed on a warming platform of a small animal physiology monitoring system (Harvard Apparatus, Holliston, MA, USA), and the front extremities were fixed with tape. A rectal thermometer was inserted to allow regulation of the heating platform to ensure a body temperature of 37°C. Thermometer and tail were fixed with tape. Mice were intubated for positive pressure ventilation (Kent Scientific, Torrington, CT, USA; 40% O2, 120 breathing cycles per minute). Isoflurane was applied at 5% until the animal stopped spontaneous respiratory movements, and then reduced to 2.0–2.5%. An infrared blood oximeter was attached to the right leg to track haemoglobin oxygen saturation, which was used to adjust the ventilation if needed. The surgical field was disinfected using Softasept N (B. Braun Melsungen). Skin and muscles were cut along the third intercostal space, and a rib spreader was introduced. The pericardium was cut, and the epicardial surface was dry-blotted using a cellulose pad. For mice undergoing ventricular cryoablation, a hexagonal metal probe (stainless steel, 2.5 mm edge-normal distance) was pre-chilled in liquid nitrogen and applied for 8–10 s on the free LV mid-wall, positioned to avoid major coronary vessels. After retraction of the probe, the time until the tissue regained deep-red color was protocoled (consistently within 5–10 s). For mice in which the ischemia-reperfusion injury was introduced, the LAD was identified and ligated with an 8-0 suture, while a narrow plastic tubing was inserted below the suture. Successful ligation was indicated by a colour change of the infarcted area from red to grey. After 30–45 min, tubing and suture were removed. For sham surgery, application of the metal probe or ligation was skipped. The rib spreader was removed, and the thorax closed using a 6-0 silk suture around the third and fourth ribs (4–5 single knots). Before final closure, any remaining air was removed from the thorax using a small cannula. Isoflurane application was stopped, and the skin was closed with a 4-0 silk suture. Once the mouse started breathing spontaneously, intubation and fixation were terminated, and the mouse was transferred to a heated and oxygenated wake-up chamber. Analgesia was maintained for 72 h post-surgery by twice-daily subcutaneous injection of 250µl of buprenorphine (10µg/mL in 154 mM NaCl; morning and late afternoon). During the night, buprenorphine was also supplied via the drinking water (10µg/mL buprenorphine (Subutex lingual tablets, Indivior) in 20 mM glucose solution).  Tissue Processing and Cell Isolation The tissue collection and slicing protocol was adapted from a previously published protocol 52,53. In brief, mice received sodium-heparin solution (16units/g body weight) by intraperitoneal injection, before being sacrificed by cervical dislocation five min later. The chests were opened, and hearts were excised. To wash out the blood from the heart, hearts were cannulated and flushed with cold 'cutting solution' (in mM): NaCl 138, NaH2PO4 0.33, KCl 5.4, MgCl2 2, HEPES 10, glucose 10, CaCl2 0.5, 2,3-butanedione 2-monoxime (BDM) 30, pH adjusted to 7.3 with 1 M NaOH at 37°C, osmolality 330 ± 10mOsm/L. Tissue blocks of ventricular free wall containing the post-cryoablation or post-ischemia-reperfusion lesion centre (identified as a region appearing mostly white), BZ and adjacent myocardium (or corresponding tissue areas from sham animals) were excised and embedded in low-melting-point agarose (4% (m/v)) at 37°C, and then put on ice. Agarose blocks were glued to the stage of a precision vibratome and cut into 300µm thick slices (60Hz cutting frequency, 1.5Hz amplitude, model 7000smz-2; Campden Instruments, Loughborough, UK). Resulting tissue slices were subjected to chemical fixation, cryopreservation, or cell isolation. For chemical fixation, slices were exposed for 30min to 4 % paraformaldehyde (PFA)-containing phosphate-buffered saline (PBS) at room temperature (RT), washed three times in PBS, and then stored in PBS at 4°C. To cryopreserve tissue, each slice was placed flat in the bottom center of a cryo-mold (Tissue-Tek, 4566), immersed in a thin layer of optimal cutting temperature compound (OCT, Tissue-Tek, 4583). To fix the orientation and keep the slice flattened, an additional cryomold of the same size was placed on top of the tissue, before snap-freezing in liquid nitrogen. Frozen slices were stored at -80°C until cryosectioning. For cell isolation, tissue slices were stored at 4°C in cutting solution and warmed to RT before initiating cell isolation. Tissue slices were transferred to 'enzymatic solution' containing (in mM): KCl 20, KH2PO4 10, MgCl2 2, Taurin 20, glucose 10, l-glutamic acid potassium salt monohydrate 100, BDM 30, pH adjusted to 7.3 with KOH at 37°C. Tissue was digested for 12min with proteinase XXIV (concentration: 0.5mg/mL; Sigma-Aldrich, St. Louis, MO, USA) and further digested with Liberase TL Research Grade (concentration: 0.23mg/mL with 5μM CaCl2; Hoffmann-La Roche, Basel, Switzerland) for up to 45 min. To remove large tissue fragments, the cell suspension was filtered through a nylon mesh (pore size: 1mm x 1mm).  NM antibody labelling and FACS sorting FACS sorting was performed using a BD FACS Symphony S6 or FACSAria™ III Cell Sorter (BD BioSciences, Franklin Lakes, NJ, USA). The same gating and collection strategy was applied across all time points post-lesioning to ensure comparable cell type ratios. For each of the collected NM sample for 10x GEM generation, isolated cells were first blocked by the TruStain FcX™ Fc blocking antibody (Biolegend, 101319) for 15 min at 4°C. Cells were labeled for 30min at 4°C in dark with fluorophore-conjugated antibodies against various surface markers, including CD45-Pacific BlueTM (Biolegend, 103125), Ter119-APC/Cy7 (Biolegend, 116223), CD31-PE (Biolegend, 102507), CD146-PE (Biolegend, 134703), CD14-FITC (Biolegend, 123307) and CD11c-APC (Biolegend, 117309), together with the Zombie-NIR viability dye (BioLegend, 423105) which labels dead cells. All antibodies were diluted at 1:100 in FACS buffer [PBS with 10% fetal bovine serum (FBS, VWR, MDTC35-016-CV) containing 1% Penicillin-Streptomycin solution (P/S, 100U/mL, Gibco, 15140122)]. When performing FACS gating, half of the cells were collected from the Zombie-NIR and Ter119-negative gate (unbiased), and another half of the cells were collected from the enrichment gates (See Supplementary Figure 1a). Cells were sorted into FACS buffer-coated 1.5 mL tubes, each with 40μL of FACS buffer with 1:1000 diluted Murine RNase Inhibitor (New England Biolabs, M0314L). After collection. Cell counting was performed. In each sample, 10,000 cells from the unbiased tube and 10,000 cells from the enrichment tube were pooled together for 10x GEM generation.  Single nucleus isolation from mouse hearts For nucleus isolation, vibratome-cut cardiac slices were placed in MACS M-tubes (Miltenyi Biotec, 130-093-236), containing 1mL of nuclear staining buffer [PBS with 1% BSA (Miltenyi Biotec, 130-091-376) and 1:500 diluted (0.2U/μL) Murine RNase Inhibitor, pre-cooled to 4°C. The tube was placed into a gentleMACS Octo dissociator (Miltenyi Biotec, 130-096-427), and then dissociated with in-built protocol “Protein_01.01 M-tube”. The M-tubes then underwent pulse-spinning, were flushed with 1mL of nuclear staining buffer, and filtered through a 40-μm cell strainer (Falcon, 1172689). The eluates were collected in 50mL Falcon tubes, which were then centrifuged for 5 min in 500g, 4°C, break = 5. Supernatants were removed, and pellets were resuspended in 500μL of nuclear staining buffer.  Nuclei antibody labelling and FACS sorting Isolated nuclei from different time points were first labelled with 1:100 diluted PCM-1 antibody (MERCK, HPA023370) in nuclear staining buffer for 5 min at 4°C (NOT on ice), 1:100 diluted fluorophore-conjugated anti-Rabbit IgG secondary antibody (Biolegend, 406419) for additional 25 min, 4°C in dark. Samples then underwent centrifugation (5min, 500g, 4°C, break = 5), followed by multiplexing with CellPlex reagents according to the manufacturer's instruction (10x Genomics, PN-1000261), resulting in time point specific labelling in each sample. After labelling, nuclei were washed with 2ml nuclear staining buffer and centrifuged (5min, 500g, 4°C, break = 5). Labelled nuclei from all time points were then pooled together in 1mL nuclear staining buffer containing 10μg/mL Hoechst 33,342 (Thermo Fisher Scientific, H3570), and underwent FACS sorting to enrich for Hoechst+ PCM-1+ nuclei. Enriched nuclei were collected in a 1.5mL tube pre-coated with nuclear staining buffer, containing 150μL of nuclear staining buffer with 1:100 diluted of Murine RNase Inhibitor. Within the collected nuclei, 10μL was stained with Trypan Blue solution (ThermoFisher, 15250061) at a ratio of 1:1, and was visually inspected under a light microscope (20x objective) to assess nuclei shape and morphology. If the majority of nuclei appeared to be intact (no visible disruption of nuclear membrane), this was followed by nuclear counting. 20,000 nuclei were used for 10x GEM generation.  Sample processing for sequencing In each sample, up to 20,000 sorted cells were loaded into GEM Chip (10x Genomics, PN-1000127) and encapsulated into emulsion droplets using the Chromium Controller (10x Genomics, Pleasanton, CA, USA) according to the manufacturer's instruction. cDNA library generation and amplification were performed with the protocol “Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (Dual Index)” according to manufacturer's instructions (10x Genomics, PN-1000268 and PN-1000190). The prepared cDNA library was then sequenced on the Nextseq2000 platform (Illumina, San Diego, CA, USA). The CellRanger v6.1.1. pipeline was used to generate a digital gene expression matrix starting from raw data. For alignment and quantification of gene expression the reference transcriptome has been built using the mouse mm10-2020-A as reference genome. De-multiplexing of CellPlex barcodes into individual samples was performed by Seurat 46. Library was performed according to the manufacter's instructions (single cell 3' v3.1 protocol, 10x Genomics). Briefly, cells/nuclei were resuspended in the maxter mix and loaded together with partitioning oil and gel beads inot the chip togenerate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to two cDNA samples, known as gene expression cDNA (GEX) and CellPlex cDNA (CMO), respectively, each contains and Illumina R1 primer sequence, unique molecular identifier (UMI) and the 10x Barcode. The pooled GEX cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and appropiate fragment size was selected with SPRIselect reagent for subsequent library construction. The CMO cDNA was directly amplifed by PCR and then was purified with SPRIselect reagent. During the library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and sample index were added.