GSM9004771: Cardiac, Heart Failure, DCM, s12; Homo sapiens; RNA-Seq
70-100 mg of Snap frozen mid-myocardium tissues samples were mechanically homogenized gently using Miltenyi gentleMACS™ Tissue Dissociator with gentleMACS M tubes using the protocol “protein_01_01” in 5 ml lysis buffer solution (5 mM CaCL2, 3mM MgAc, 2mM EDTA, 0.5mM EGTA, 10mM Tris-HCL in water supplemented with 2mg/ml Actinomycin D, 1:1000 protease inhibitor cocktail, and 40 U/µl RNase OUT), then incubated on ice for 15 min with extra 5 ml of the same solution supplemented with 0.1% NP-40. Lysate was then gently filtered through a 30 μm filter into 15 ml conical tube, followed by rinsing the filter once with 2 ml lysis buffer supplemented with 0.1% NP-40. Nuclei were then centrifuged at 1000g for 5 min at 4 °C. Nuclei pellet was then resuspended in 2 ml of sucrose buffer solution (1 M sucrose, 3mM MgAc, and 10 mM Tris-HCL supplemented with 2mg/ml Actinomycin D, 1:1000 protease inhibitor cocktail, 40 U/µl RNase OUT, and 2µl/ml WGA) and layed over another 2 ml sucrose solution in 5 ml canonical tube. Centrifugation was repeated according to the above parameters. Supernatant was then removed and nuclei were resuspended in 400 μl nuclei wash buffer (PBS with 40 U/µl RNase OUT, 15 µl/ml UltraPure BSA,and 2µl/ml WGA) and centrifuged at 650g for 6 min at 4 °C. Another washing step repeated similar to the previous step with centrifugation speed of 500g, and a third wash was performed with 1 µl/ml DAPI and incubated for 10 min in 1.5 ml Eppendorf tube before centrifuging again at 500g speed. The pellet was then resuspended in 300 µl of PBS and filtered in FACS tubes with 35 µl strained. Nuclei were sorted based on size and DAPI positive staining into 40 µl PBS + 15 µl/ml UltraPure BSA using 100-µM nozzle, and collected in 1.5 ml tubes. Single sorted nuclei were counted by LUNA automated counter in duplicates, then nuclei were centrifuged again and were gently resuspended to a target concentration of 1,000 nuclei/µl and loaded on the Chromium Controller (10X Genomics) with a targeted cell or nuclei recovery of 6,000–10,000 per reaction. 3′ gene expression libraries were prepared according to the manufacturer's instructions of the v3 Chromium Single Cell Reagent Kits (10X Genomics). Quality control of cDNA and final libraries was done using Bioanalyzer High Sensitivity DNA Analysis (Agilent) and libraries were sequenced using NovaSeq 6000 (Illumina) at Genomics Core facility at KU Leuven targeting 80,000 read pairs nucleus. Nuclei were aligned to the human GRCh38-3.0.0 transcriptome with “Include-introns= TRUE” using the CellRanger- 6.0.1 software (10X Genomics) according to the 10X Genomics' instructions.