Illumina NovaSeq 6000 paired end sequencing; DNMT3A clonal hematopoiesis-driver mutations induce cardiac fibrosis by paracrine activation of fibroblasts
DNMT3A clonal hematopoiesis-driver mutations induce cardiac fibrosis by paracrine activation of fibroblasts
DNMT3AR882H knock-in (KI) mice were generated by the Mouse Biology Program (MBP) of the University of California (Davis, CA) as previously described34. The DNMT3Afl-R882H-fl mice were crossed to B6.Cg-Tg(Mx1-cre)1Cgn/J mice (referred to as Mx1-Cre)57 or to tamoxifen-inducible transgenic B6.129-Gt(ROSA)26Sortm1(cre/ERT2)Tyj/J mice (referred to as R26-CreERT2 Cre recombinase - estrogen receptor T2)58. C57BL/6N (CD45.2 or Ly5.2) and B6.SJL-PtprcaPepcb/BoyJ (CD45.1 or Ly5.1) mice were obtained from Charles River Laboratory. Primary bone marrow cells of age-matched Mx1-Cre+/DNMT3Afl-R882H-fl and Mx1-Cre-/DNMT3Afl-R882H-fl donor mice (Ly5.2) were collected between 14 to 28 days after the last pI:pC  injection (Amersham; 400μg/mouse intraperitoneally for 3 nonconsecutive days) to ensure that signaling activation and cytotoxic effects mediated by pI:pC were minimized. To validate excision efficiency, genomic DNA from blood or harvested cells was subjected to PCR. Recipient mice were lethally (7.5 Gy total body irradiation) irradiated using X-Ray Irradiation System MultuRad 160 (Faxitron Bioptics LLC, USA). Recipient mice were maintained on Cyprofloxacin drinking water (40/mg/kg, Fresenius Kabi Deutschland GmbH, Bad Homburg) for one week before and two weeks after transplantation and allowed to engraft for 6 weeks, before being used for analysis. For transplantation, donor bone marrow cells from primary +/m (Ly5.2+) or wild-type +/+ (Ly5.2+) mice were transplanted into lethally irradiated Ly5.1 (B6.SJL-Ptprca Pepcb/BoyJ; Charles River Laboratory) recipients with a total of 2.5 x 105 cells per recipient. Peripheral blood engraftment was assessed by flow cytometry 6 weeks post transplantation. Six weeks after reconstitution of the bone marrow, myocardial infarction was induced by permanent ligation of the left anterior descending artery in transplanted recipients. In brief, anesthesia was induced with isoflurane (4%/800 ml O2/min) and maintained by endotracheal ventilation (2–3%/800 ml O2/min). Thoracotomy was performed in the fourth left intercostal space. The left ventricle was exposed, and the left coronary artery was permanently occluded. Chest and skin were closed, and anesthesia was terminated. Animals were extubated when breathing was restored. Initial myocardial injury was evaluated by measuring cardiac Troponin T levels in plasma 24 h after induction of myocardial infarction.  For mouse single-cell nuclei (sn) and single-cell (sc) RNA sequencing we used a transplantation model with tamoxifen-inducible Rosa26CreERT2:DNMT3A-R882H (Cre recombinase - estrogen receptor T2; known as R26-CreERT2 strain) intercross mice as donors. These mice do not require pI:pC treatment and are independent of intrinsic inflammatory IFN-response to induce DNMT3A-R882H expression within the hematopoietic compartment34. We generated complete “chimeras” mice by transplanting of non-induced Rosa26CreERT2:DNMT3A-R882H (Ly5.1+) hematopoietic cells (either (+/+) or (+/m)) into Ly5.1/2 recipients. 6 weeks after BM cells transplantation, recipients were subjected to myocardial infarction as described above. Dissolved tamoxifen (Sigma-Aldrich) in corn oil was administered via intraperitoneal injection (1mg/mouse/day) once every 24 hours for a total of 10 consecutive days. The injections started 28 days after the induction of the myocardial infarction. The mice were used at day 74-76 post LAD. Cardiac tissues were thawed on ice and cut into small pieces. Minced tissue was pre-digested with a 5-ml enzyme solution of collagenase (2,500 U, Thermo Fisher Scientific) in HBSS+/+ (Gibco) for 10 min at 37 °C in a water bath. After centrifugation at 500g and 4 °C for 5 min, the supernatant was discarded, and nuclei were isolated after cell disruption with a glass dounce homogenizer (five strokes with a loose pestle and ten strokes with a tight pestle). After filtering (20-µm strainer, pluriSelect), the suspension was centrifuged at 1,000g and 4 °C for 6 min and resuspended in 500 µl staining buffer containing 1% BSA (Sigma-Aldrich), 5 nM MgCl2 (Sigma-Aldrich), 1 mM EDTA (Gibco), 1 mM EGTA (Gibco), 0.2 U µl−1 RNasin Plus Inhibitor (Promega) and 0.1 µg ml−1 Hoechst (Life Technologies) in Dulbecco´s phosphaste buffered saline (DPBS). Hoechst-positive nuclei were separated from cell debris by using the FACSAria Fusion instrument (BD Biosciences) and sorted into staining buffer without Hoechst at 4 °C. Cellular suspensions were loaded on a 10X Chromium Controller (10X Genomics) according to manufacturer's protocol based on the 10X Genomics proprietary technology. Single-cell RNA-Seq libraries were prepared using Chromium Single Cell 3′ Reagent Kit, v3 (10X Genomics) according to manufacturer's protocol. Briefly, the initial step consisted in performing an emulsion capture where individual cells were isolated into droplets together with gel beads coated with unique primers bearing 10X cell barcodes, UMI (unique molecular identifiers) and poly(dT) sequences. Reverse transcription reactions were engaged to generate barcoded full-length cDNA followed by the disruption of emulsions using the recovery agent and cDNA clean up with DynaBeads MyOne Silane Beads (ThermoFisher). Bulk cDNA was amplified using a Biometra Thermocycler TProfessional Basic Gradient with 96-Well Sample Block (98°C for 3 minutes; cycled 14×: 98°C for 15s, 67°C for 20s, and 72°C for 1 minute; 72°C for 1 minute; held at 4°C). Amplified cDNA product was cleaned with the SPRIselect Reagent Kit (Beckman Coulter). Indexed sequencing libraries were constructed using the reagents from the Chromium Single Cell 3′ v3 Reagent Kit, as follows: fragmentation, end repair and A-tailing; size selection with SPRIselect; adaptor ligation; post-ligation cleanup with SPRIselect; sample index PCR and cleanup with SPRI select beads. Library quantification and quality assessment was performed using Bioanalyzer Agilent 2100 using a High Sensitivity DNA chip (Agilent Genomics). Indexed libraries were equimolarly pooled and sequenced on an Illumina NovaSeq 6000.