GSM9650164: Neonatal rat cardiomyocyte, GFP, rabbit YAP antibody; Rattus norvegicus; OTHER
RNA was harvested using RNeasy Mini Plus Kit (Qiagen). 1.3 μg of total RNA was used for the construction of sequencing libraries. For CUT&RUN, DNA was released by incubation with 1/100 volume of 10% SDS and 1/80 volume of 2.5 mg/mL Proteinase K (Thermo Fisher) for at least 1 hour at 55 °C. DNA was then purified by phenol/chloroform/isoamyl alcohol extraction, followed by ethanol precipitation with glycogen, and resuspended in nuclease-free water. For RNA seq, messenger RNA (mRNA) was purified from total RNA using poly-T oligo-attached magnetic beads. Following fragmentation, first-strand cDNA was synthesized using random hexamer primers, and second-strand cDNA synthesis was performed using dUTP instead of dTTP to maintain strand specificity. Directional libraries were prepared through a series of steps, including end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification. Library quality was assessed using Qubit fluorometry, real-time PCR, and fragment size distribution by Bioanalyzer. Libraries were pooled based on effective concentration and desired sequencing depth, then subjected to Illumina sequencing.  For CUT&RUN, DNA was released by incubation with 1/100 volume of 10% SDS and 1/80 volume of 2.5 mg/mL Proteinase K (Thermo Fisher) for at least 1 hour at 55 °C. DNA was then purified by phenol/chloroform/isoamyl alcohol extraction, followed by ethanol precipitation with glycogen, and resuspended in nuclease-free water. For CUT&RUN, Sequencing libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) with minor modifications. End repair was carried out for 30 minutes at 20 °C, followed by A-tailing at 65 °C for 30 minutes. After adapter ligation (30 minutes at 20 °C), DNA fragments were purified using 1.8× SPRIselect beads (Beckman Coulter), then amplified via PCR for 17 cycles using NEBNext Ultra II Q5 Master Mix (New England Biolabs). Final libraries were purified with 0.9× SPRIselect beads and quantified with the Qubit dsDNA HS Assay Kit (Agilent Technologies).