GSM9419873: Rat, cardiomyocytes, Sham2, anti-ERRβ (Input); Rattus norvegicus; OTHER
Excised hearts were rinsed in ice-cold 1× PBS to remove residual blood and adipose tissue, and the myocardium was minced into ~1 mm³ pieces on ice. For each preparation, 0.1-0.3 g of minced tissue was transferred into 3 mL pre-chilled Buffer A and homogenized in a glass Dounce homogenizer using a loose-clearance pestle. When indicated to suppress RNase activity, 1 mL citrate solution was added during homogenization. The tissue suspension was triturated once with the loose pestle, incubated on ice for 5 min, and further triturated 5–10 strokes. After an additional 5 min on ice, the sample was triturated 3–5 strokes with the loose pestle and then 5 strokes with the tight-clearance pestle. The homogenate was filtered through a 100 µm cell strainer, and the strainer was rinsed sequentially with 1 mL and 750 µL Buffer A to maximize nuclei recovery. Triton X-100 was then added to a final concentration of 0.5% (v/v), and the suspension was further homogenized by trituration (20-50 strokes). The lysate was passed through a 35 µm cell strainer and centrifuged at 500g for 5 min at 4°C. The supernatant was discarded, and the pellet was resuspended in 1 mL citrate solution, followed by centrifugation at 500g for 5 min at 4°C. The pellet was subsequently centrifuged at 3,000g for 5 min at 4°C, resuspended in 1 mL Buffer B and centrifuged again, and the resulting pellet was resuspended in 200 µL Buffer B. A final clarification spin was performed at 20,000g for 3 min at 4°C. Nuclei were assessed by trypan blue staining and quantified using a Countess automated cell counter. All procedures were carried out on ice or at 4°C unless otherwise specified. CUT&Tag (Cleavage Under Targets and Tagmentation) libraries were constructed using the Vazyme CUT&Tag Library Prep Kit for Illumina (Vazyme, Cat# TD903) following the manufacturer's instructions with minor modifications. Cell Preparation and Bead Binding Briefly, approximately 1 X 105 cells per sample were harvested and washed twice with Wash Buffer. Concanavalin A-coated magnetic beads (Vazyme) were activated and added to the cell suspension, followed by incubation at room temperature for 10 min to allow cell-bead binding. Primary and Secondary Antibody Incubation The bead-bound cells were resuspended in Antibody Binding Buffer containing the specific primary antibody ERRβ and incubated at 4 ℃ overnight with gentle agitation. After washing to remove unbound antibodies, the cells were incubated with a secondary antibody (Goat Anti-Mouse IgG) in Dig-Wash Buffer at room temperature for 1 hour. Tagmentation and DNA Extraction The cell-bead complexes were washed three times to remove residual secondary antibodies and then incubated with pA-Tn5 (protein A-Tn5) adapter complex at room temperature for 1 hour. To activate tagmentation, the samples were resuspended in Tagmentation Buffer and incubated at 37℃ for 1 hour. The reaction was terminated by adding EDTA, SDS, and Proteinase K, followed by incubation at 55℃ for 1 hour to digest proteins and release DNA fragments. Genomic DNA was subsequently extracted via phenol-chloroform or using the DNA Clean Beads provided in the kit. Library Amplification and Purification The extracted DNA was used as a template for PCR amplification to add Illumina-compatible sequencing adapters. The PCR program was set as follows: initial extension at 72℃ for 3 min; denaturation at 95℃ for 3 min; followed by 12 cycles of 98℃ for 10 s and 60℃ for 5 s; and a final extension at 72℃ for 1 min. The resulting libraries were purified using VAHTS DNA Clean Beads (Vazyme, Cat# N411) at a ratio of 2.0 X to remove primer dimers.