GSM7051201: P10_Sham_7D_RZ_rep2; Mus musculus; ChIP-Seq
The heart tissues were roughly divided into three zones (infarct zone, border zone and remote zone) and then digested with collagenase type II enzyme at 37°C for 30 min to generate single-cell suspensions. F4/80/CD45+ cardiac macrophages were collected by FACS sorting and fixed with 0.25% formaldehyde for 5 min on ice followed by methyl alcohol fixation. Before single-cell CoBATCH experiments, the cells in methyl alcohol were washed with 0.1% BSA/PBS for three times. Custom library preparation was performed by two steps of PCR amplification. The first step was performed by addition of 0.5 μL 50 μM Truseq connector primer (Connector primer F:5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCGTCGGCAGCGTCTCCACGC-3′,  Connector primer R:5′-GACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTCTCGTGGGCTCGGCTGTCCCTGT-3′), 25 μL 2X Taq master mix and 16.5 μL 1mM MgCI2 to the 3 μL CoBATCH cell lysis in each tube and the reaction was set up by incubation at 72°C for 5 min, 95°C for 5 min, 13-16 cycles of amplification (95°C for 30 s, 63°C for 30 s, 72°C for 1 min), and final 72°C extension for 5 min. The PCR enrichment was directly performed in the same tube as described above. Then 0.4 μL Exo1 was added and incubated at 37°C for 1 h followed by inactivation at 72°C for 20 min. The second PCR enrichment was performed by adding 0.7 μL 10 μM Truseq i5 primer (5′- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC-3′), 0.7 μL 25 μM i7 index primer (5′-CAAGCAGAAGACGGCATACGAGAT[i7]GTCTCGTGGGCTCGG-3′), 20 μL 2X Taq master mix and 8.6 μL 1 mM MgCI2 followed by incubation at 95°C for 5 min, 5-7 cycles of  95°C for 30 s, 63°C for 30 s, 72°C for 1 min, and final 72°C extension for 5 min. Finally, the PCR products were purified and selected by SPRI beads for 200-1000 bp.