F123 (F1 Mus musculus castaneus x S129/SvJae mouse ESC line)
chip antibody
none
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
2 million fixed F123 cells were thawed on ice, resuspend in hypotonic lysis buffer (20 mM HEPES, pH 8.0, 10 mM KCl, 1 mM EDTA, 10% glycerol) with proteinase inhibitors and rotate at 4 °C for 15 minutes. The nuclei were then washed once with hypotonic lysis buffer with proteinase inhibitors and resuspend in 130 μL RIPA buffer (10 mM Tris, pH 8.0, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate) with proteinase inhibitors. After incubation on ice for 10 minutes, the nuclei were sheared using Covaris M220 with following setting: power, 75 W; duty factor, 10%; cycle per burst, 200; time, 10 minutes; temp, 7 °C. The cell lysate was cleared by centrifugation at 15,000×g for 20 min and supernatant was collected. The clear cell lysate was precleared with Protein G Sepharose beads (GE Healthcare) and for 3 hours at 4 °C with slow rotation. ~5% of precleared cell lysate was saved as input control. The rest of the lysate was mixed with 2.5 µg of H3K4me3 (04-745, Millipore) antibody and rotate at 4 °C for at least 12 hours. On the next day, 0.5% BSA-blocked Protein G Sepharose beads (prepared one day ahead) were added and rotated for another 3 hours at 4 °C. The beads were collected by centrifugation at 400×g for 1 min and then washed with RIPA buffer three times, high-salt RIPA buffer (10 mM Tris, pH 8.0, 300 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate) twice, LiCl buffer (10 mM Tris, pH 8.0, 250 mM LiCl, 1 mM EDTA, 0.5% IGEPAL CA-630, 0.1% sodium deoxycholate) once, TE buffer (10 mM Tris, pH 8.0, 0.1 mM EDTA) twice. Washed beads were treated with 10 µg Rnase A in extraction buffer (10 mM Tris, pH 8.0, 350 mM NaCl, 0.1 mM EDTA, 1% SDS) for 1 hours at 37 °C, followed by reverse crosslinking in the presence of proteinase K (20 µg) overnight at 65 °C. After reverse crosslink the DNA was purified by Zymo DNA Clean&Concentrator. 10-100 ng ChIP DNA or input DNA was first end repaired at 20 °C for 30 minutes in 1×T4 DNA ligase buffer (NEB) with 0.5mM dNTP mix, 3U T4 DNA polymerase (NEB), 2.5U Klenow fragment (NEB) and 10U T4 PNK (NEB). The repaired DNA was then purified by Zymo DNA Clean&Concentrator and adenylated at 37 °C for 30 minutes in 1×NEBbuffer 2 (NEB) with 0.4mM dATP, 10U Klenow fragment (3'-5' exo-) (NEB). The adenylated was purified by Zymo DNA Clean&Concentrator and ligated to the adapters (Illumina, TruSeq, 0.1 μL per 100ng DNA) at 16 °C for overnight in 1×T4 DNA ligase buffer (NEB) with 400U T4 DNA ligase. After purification with Zymo DNA Clean&Concentrator, DNA was amplified with KAPA HiFi HotStart ReadyMix PCR Kit for 12 cycles according to the manufacturer's instructions. The amplified libraries were purified with Ampure Beads to extract fragments between 200-600bp for sequencing.