title: Sample 1
organism: Mus musculus
description: Protocols: 12 weeks and 18 months old male C57Bl/6J wildtype mice purchased from Janvier (Le Genest Saint-Isle, France) were used in this study. The following transgenic mice were used: Rgs5tm1a(EUCOMM)Wtsi (MGI ID:4432511, Rgs5fl/fl)9 , Pdgfrb-CreERT212 . Pericyte-specific Rgs5 knockout (Rgs5 PC) was generated by breeding Rgs5fl/fl mice with Pdgfrb-CreERT2 mice. Rgs5 deletion was induced in mice aged between 8 and 10 weeks by tamoxifen intraperitoneal injection (20 mg/ml), performed for five consecutive days (100 µl/mouse/day). Homozygous Rgs5fl/fl mice were used for animal experiments. We compared animals carrying one copy of Pdgfrb-CreERT2 with Cre-negative littermate controls. Experiments were performed in both females and males. The transgenic mice experiments were approved by the Regierungspraesidium Darmstadt under application number FU/2015. hearts were perfused with 1X DPBS (Gibco) and directly cut into pieces. Tissue was minced in pre-filtered homogenization buffer containing 250 mM sucrose (Sigma-Aldrich), 25 mM KCl (Invitrogen), 5 mM MgCl2 (Invitrogen), 10 mM Tris buffer pH 8.0 (Invitrogen), 1 mM DTT (Thermo-Fisher Scientific), 1X protease inhibitor (Roche), 0.6 U/l Ambion RNase inhibitor (Thermo-Fisher Scientific), 0.1% Triton (Sigma-Aldrich) in ultra-pure DNase/RNase-free distilled water (Thermo-Fisher Scientific). Nuclei were isolated after cell disruption with a glass dounce homogenizer (ten strokes with a loose pestle and five strokes with a tight pestle). The homogenized solution was then filtered onto a 40 m cell strainer. The filtering was repeated onto a 20 m cell strainer, before centrifuging the cell suspension at 500 g at 4°C for 8 min. After careful removal of the supernatant, the cell pellet was resuspended in sorting buffer containing 1% BSA (Sigma-Aldrich), 0.2 U/l Ambion RNase inhibitor (Thermo-Fisher Scientific), 1 mM DTT (Thermo- Fisher Scientific) in DPBS (Gibco). 7AAD (Biolegend) positive nuclei were separated from cell debris using the FACSAria Fusion instrument (BD Biosciences) and sorted into collection buffer containing 1% BSA (Sigma-Aldrich), 1 U/l Ambion RNase inhibitor (Thermo-Fisher Scientific), 1 mM DTT (Thermo-Fisher Scientific) in DPBS (Gibco). Sorted nuclei were washed twice with 0.04% BSA (Sigma-Aldrich) in DPBS (Gibco). Single-nucleus RNA-sequencing library preparation: sorted nuclei were subjected to single-cell. RNA sequencing using the 10X Genomics platform and the chromium Next GEM Single Cell v3.1 workflow. Briefly, 20000 nuclei were loaded on a Chip G (10X Genomics) and run in the Chromium Controller to capture and barcode poly-adenylated transcripts of single nuclei in Gel-Beads-in emulsion. Following reverse transcription, the full-length cDNA was amplified and purified with Dynabeads MyONE SILANE (10X Genomics). Single-cell gene expression libraries were obtained by enzymatic fragmentation, ligation of sequencing adapters and attachment of i5 and i7 sample indexes. A final quality control was performed on an Agilent High Sensitivity Chip using the Agilent Bioanalyzer 2000 system.