GSM6632635: hESC_H9_d4_BPK25_IP_TET2_rep3; Homo sapiens; ChIP-Seq
Lysates were clarified from sonicated nuclei and protein-DNA complexes were isolated with antibody (CTCF (abcam, Cat. #ab128873), MBD3 (abcam, Cat. #ab157464) and TET1 (GeneTex 125888)). Sequencing libraries were made following the protocol of NEB Next ChIP-Seq DNA Sample Prep Kit with minor modifications. To perform end repair of DNA fragments, 12 μL mixture (5µL NEB T4 DNA ligase buffer with 10mM ATP, 2µL dNTPs mix 10mM each, 2µL T4 DNA polymerase 3u/µL, 1µL Klenow DNA polymerase 5u/µL, 2µL T4 PNK 10u/µL) was added to 38 μL of IP DNA and input DNA (20 ng in 38 μL) and incubated for 30 min at 20 °C. The DNA was then purified using VAHTS® DNA Clean Beads (N411-01, Vazyme) and eluted in 41 μL H2O, following the manufacturer's instructions. To prepare the DNA fragments for ligation to adapters by adding an “A” base to the 3' end, 9 μL mix (5µL NEB Buffer 2, 1µL dATP 100mM, 3µL Klenow 3'-5' exo minus 5u/µL) was added to 41 μL DNA from previous step and incubated for 30 min at 37 °C. After “A” base addition, the DNA was then purified using VAHTS® DNA Clean Beads (N411-01, Vazyme) and eluted in 42 μL H2O. To ligate adapters to DNA fragments, 8 µL reaction mixture (1µL adapter mix (1uM) (1000005282, MGI), 5µL NEB T4 DNA ligase buffer, 2µL NEB T4 DNA ligase) was added to 42 μL DNA from the previous step and incubated at 16 °C overnight. Size selection (200–500 bp) was achieved using a 1.5% agarose gel and NEB Monarch DNA Gel Extraction Kit (T1020S), and the DNA was eluted in 20 μL H2O. Finally, the libraries were amplified by 18 PCR cycles using Phusion High-Fidelity DNA Polymerases (M0530L, NEB) with the DNA from size selection and purified as size selection. The libraries were sequenced with an MGIseq2000 instrument (BGI, Shenzhen) using a paired-end scheme (PE100).