title: GSM6652440: dpi7_CUT&Tag_H3R27ac_FB:dnAP-1_replicate1 [ROSL_T_1]; Danio rerio; OTHER
library_construction_protocol: freshly isolated noncardiomyocytes nonCMs from injury area of adult zebrafish hearts were used as input to immobilize on concanavalin-coated magnetic beads (Bangs Laboratories, cat. no. BP531). Beads are incubated with primary antibody H3K27ac (Abcam, ab4729) or IgG (Abcam, ab172730) overnight at 4°C, followed by incubation with a secondary antibody (Anti-Rabbit Secondary Antibody, EpiCypher, 13-0047) for 1h at room temperature. Beads are washed using Dig-wash buffer and incubated with pA-Tn5 mix (EpiCypher, 15-1017) and then resuspended with Tagmentation buffer, gently mixed, and incubated for 1 h at 37°C for adapter tagmentation reaction. To stop tagmentation and solubilize DNA fragments by de-crosslinking, 10 μl of 0.5 M EDTA, 3 μl of 10% SDS, and 2.5 μl of Proteinase K (20 mg/mL) are added to each sample and mixed thoroughly. The reaction tubes are then incubated at 55°C for 1h. After de-crosslinking, DNA is purified by Phenol-Chloroform-Isoamyl alcohol (25:24:1) extraction and Chloroform extraction followed by ethanol precipitation. Air-dried purified DNA is dissolved in 30 μl of 10 mM Tris-HCl pH 8.0 containing 1 mM EDTA and 25 μg/ml RNase A. DNA fragments are amplified and indexed by uniquely barcoded i5 and i7 primers with the NEBNext High-Fidelity 2× PCR Master Mix (New England Biolabs, M0541S). The CUT&Tag libraries are purified using AMPure beads and are analyzed using an Agilent 2100 Bioanalyzer Instrument and High Sensitivity DNA Chips to detect the size distribution of libraries.