T-2 (CRX1177861; CRA018692)
Initially,  cells were collected for each immunoprecipitation. For chromatin digestion, cells were mixed with 20 uL Nuclear Isolation Buffer and 30 uL MNase Master Mix (10 U/mL) at 25 deg C for 7 minutes. The digestion process was halted by adding 5.5 uL of 0.1 M EDTA and 5.5 uL of a solution containing 1% Triton X-100 and 1% deoxycholate. Antibody-coated bead complexes were prepared by incubating 11 uL Dynabeads Protein G (Invitrogen, 10003D) with 1 ug of H3K4me3 antibodies in Complete Immunoprecipitation Buffer for 6 hours at 4 deg C, then added to the samples. After overnight incubation at 4 deg C, the samples were washed twice with Low Salt and High Salt Wash Buffers, respectively. Chromatin was eluted in 100 uL ChIP Elution Buffer at 65 deg C for 2 hours. DNA, with a 2 uL spike-in, was extracted using phenol-chloroform-isoamyl alcohol (25:24:1) (Sigma, 77617) and prepared for library construction using the KAPA HyperPrep Kit (Roche, KK8504) according to the manufacturer's instructions. Sequencing was performed using paired-end 150-bp reads on the Illumina NovaSeq 6000 platform.