title: GSM4715075: TAC_JQ1_bulkRNAseq_CMs_Rep1; Mus musculus; RNA-Seq
library_construction_protocol: Briefly, after proper anesthesia level was reached, thoracotomy was performed, and mouse heart was isolated. The isolated heart was cannulated and perfused with perfusion buffer (120.4 mM NaCl, 14.7 mM KCl, 0.6 mM KH2PO4, 0.6 mM Na2HPO4, 1.2 mM MgSO4, 10 mM Na-HEPES, 4.6 mM NaHCO3, 30 mM taurine, 10 mM 2,3-butanedione monoxime, and 5.5 mM glucose, pH 7.0) in a Langendorff perfusion system (Radnoti 120108EZ) for 5-10 minutes at 37 °C. The cannulated heart was then digested by digestion buffer (perfusion buffer with 300 units/mL collagenase II (Worthington Biochemical) and 50 μM CaCl2) for about 10 min at 37 °C. At the end of digestion, the atria and great vessels were removed and the ventricular tissue was transferred to and gently teased into small pieces in stop buffer (perfusion buffer with 10% fetal bovine serum) at 37 °C. After gently pipetting, cell suspension was passed through a 250uM strainer in a falcon tube and then at 30xg for 3 minutes at room temperature (RT). Then, the supernatant - containing most of the non-cardiomyocytes (CMs) - was divided from the pellet (containing the CM fraction). The non-CM fraction was centrifuged again at 30xg for 3 minutes at RT and the supernatant kept. The supernatant was then filtered with a cell strainer (70um) and finally centrifuged at 400xg for 3 minutes at RT for eliminating debris. The non-CM pellet was finally resuspended in 1mL cold PBS 0.5% BSA. 30k cells were counted with trypan blue using a hemocytometer and then used for subsequent 10X Genomics Chromium single cell RNAseq preparation. For single cell ATAC, 500k isolated and purified non-CMs were resuspended in 100uL lysis buffer (Tris-HCl 10mM pH 7.4, NaCl 10mM, MgCl2 3mM, Tween-20 0.1%, P40 0.1%, Digitonin 0.01%, BSA 1% in Nuclease-free water), pipetted 10 times and kept on ice for 5 minutes. Nuclei were then washed 1mL 1X PBS with 1% BSA and then centrifuged at for 5 minutes at 4C. The nuclei pellet was resuspended in 1mL 1X PBS with 1% BSA and filtered with a 10uM strainer (pluriSelect #43-50010-00). Nuclei were then counted with DAPI using a hemocytometer and finally 30k non-CM nuclei used for subsequent 10X Genomics Chromium single cell ATACseq preparation. The CM-fraction was, after the first centrifugation, centrifugated again at 30xg for 3 minutes at RT in stopping buffer and the supernatant was discarded. The CM pellet was finally centrifuged at 400xg for 3 minutes in stopping buffer at RT and after discarding the supernatant, the cell pellet was lysed in Qiazol (miRNeasy kit - Qiagen) for subsequent RNA extraction. Total RNA from CMs was extracted using miRNeasy kit (Qiagen) according to the manufacturer's instructions and quantified with Nanodrop (Thermo scientific). After RNA quality control with bioanalyzer Agilent 2100 (Agilent Technologies), Paired-end Poly(A)-enriched RNA libraries were prepared with the ovation RNA-seq Universal kit (NuGEN; strand specific) from the Gladstone Genomic core for 9 samples: Sham (x3), TAC-Veh (x3) and TAC-JQ1 (x3). High-throughput sequencing was done using a PE75 run on a NextSeq 500 instrument (Illumina).
attributes.tag: GEO Accession
attributes.value: GSM4715075