GSM7949969: Pre_PS_H3K4me1; Mus musculus; ChIP-Seq
The embryos were microdissected and digested with Collagenase I dissociation buffer at 37°C for 15-60 min to generate single-cell suspension. The dissociated single cells were collected by centrifugation at 600 g for 3 min. After incubation with Con-A beads, the cell-bead mixture was fixed with methyl alcohol and incubate on ice for 5 min. Custom library preparation was performed by two steps of PCR amplification. The first step was performed by addition of 0.5 μL 25 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.