title: GSM8033703: D2_H3K4me3_1; Mus musculus; OTHER
library_construction_protocol: To isolate macrophages, calf muscle from the ischemic limb was collected and digested in 2 mg/ml Collagenase IV (Thermo Fisher Scientific, 17104019) / Dispase II (Sigma-Aldrich, D4693-1G) for 1 h at 37°C. After filtration and washing steps, red blood cells were removed with ACK Lysis buffer (Gibco, A1049201).  CD45+CD11b+F4/80+CD64+ macrophages were stained and sorted (Sony Cell sorter SH800S) . The used antibodies were the following: PE anti-mouse CD45 (Biolegend [30-F11], 103106), PerCP/Cy5.5 anti-mouse/human CD11b (Biolegend [M1/70], 101228), Alexa Fluor® 488 anti-F4/80 mouse Monoclonal Antibody (Biolegend [clone: 30-F11], 103122), APC anti-CD64 mouse Monoclonal Antibody (Biolegend [clone: X54-5/7.1], 139306).  CUT&Tag was performed as described earlier starting from nuclei (Galle et al., 2022). Nuclei were isolated from the sorted macrophages. In short, the macrophages were centrifuged for 5 min at 4°C, 500 rpm, supernatant was removed, and the cells were lysed on ice in 1 mL of nucleus extraction buffer (1× prelysis buffer from the EpiGentek EpiQuick Total Histone Extraction Kit, OP-0006-100). To stop the lysis reaction, 1 mL of PBS+1%BSA was added, and nuclei were collected through centrifugation for 5 min at 4°C, 500 rpm. The supernatant was removed, the nuclei were resuspended in PBS+1% BSA, and a sample was visually inspected for viability, purity, and abundance of nuclei under the microscope. Next, CUT&Tag libraries were created according to the published CUT&Tag protocol [29] for nuclei. All buffers were supplemented with 5 mM sodium-butyrate (Sigma, 303410) and 1X complete protease inhibitor (Merck, 1187358000). Protein lo-bind tubes (Eppendorf, EP0030108116) were used to reduce sample loss. Antibodies against H3K18la (PTM-Bio, PTM-1406), H3K4me3 (Abcam, ab8580), H3K27me3 (Cell Signaling Technology, C36B11) and H3K27ac (Abcam, ab4729) were used in this study. PCR was performed using Kapa HiFi plus dNTPs (Roche Diagnostics, #07958846001) with the following PCR settings: 72˚C 5 minutes, 98˚C 30 seconds, 15 cycles of 98˚C 10 seconds, 63˚C 30 seconds, 72˚C final extension for 1 minute, hold at 4˚C. Libraries were indexed using Nextera Indexes, and 150-bp paired-end sequencing was performed on Illumina Novaseq instruments.