SKMEL2 Control ChIP Rep 3
SKMEL2 cells stably expressing 3HA-RFP, 3HA-AHR-RFP and 3HA-ARNT-RFP were cultured in 15 cm dishes separately. For each group, a total of 410 cells were harvested and resuspended in 2 mL serum-free DMEM. For cross-linking, 1% formaldehyde solution (prepared freshly) was added, and cells were incubated at room temperature (RT) for 8 min. Cross-linking was quenched by adding 0.125 M glycine and incubating at RT for 5 min. Cells were then washed once with ice cold PBS. After fixation, pellets were flash frozen and stored at -80C or processed immediately for sonication. Each pellet was lysed in lysis buffer (0.1% SDS, 1% Triton X-100, 0.1% Na-Deoxycholate, 0.25% sarcosyl, 50 mM HEPES-KOH pH7.5, 1 mM EDTA, 140 mM NaCl, 1 protease inhibitor cocktail). Samples were incubated on ice for 10-15 min, then aliquoted into thin-walled 0.5 mL PCR tubes. Sonication was performed using a Qsonica Q800R (50% amplitude, 30 s on / 30 s off, for 20 cycles) to achieve a mean DNA fragment size of 300 bp. Post-sonication, samples were centrifuged at 12,000g for 5 min at 4C, and the supernatant was collected and kept on ice or stored at 80C. Samples were diluted 1:10 in dilution buffer (1% Triton X-100, 140 mM NaCl, 50 mM HEPES-KOH pH7.5 and 1 mM EDTA) supplemented with 5 M NaCl to reach 150 mM NaCl, and incubated rotating with antibody and Protein A/G magnetic beads (Invitrogen), to bind the anti-HA antibody (Proteintech #66006-2-Ig) and associated chromatin, for 3 h at 4C. After washing seven times with high salt buffer (0.1% SDS,1%Triton X-100, 20 mM Tris pH7.9, 2 mM EDTA, 500 mM NaCl) and twice with TE buffer (10 mM Tris, 1 mM EDTA, pH8.0), samples were eluted from the beads for 15 min at 65C in elution buffer (1% SDS, 200 mM NaCl). Eluates were treated with RNase A at 37C for 30 min and reverse cross-linked overnight by heating at 65C with Proteinase K. Samples were extracted with an equal volume of phenol:chloroform, vortexed, centrifuged at 12,000g for 10 min. The aqueous phase (~400 L) was transferred to a new tube and mixed with 1.6 L GlycoBlue (250), 40 L 3 M sodium acetate (pH 5.2), and 800 L ice-cold ethanol. DNA was precipitated at 20C overnight. Samples were centrifuged at 12,000g for 15 min at 4C, washed twice with 75% ethanol, and resuspended in 20 L nuclease-free water. The ChIP library for each sample was then prepared using VAHTS Universal Pro DNA Library Prep Kit for lllumina (Vazyme #ND608) with VAHTS Multiplex Oligos Set 4 for Illumina (Vazyme #N321) and VAHTS DNA Clean Beads (Vazyme #N411) followed by Illumina sequencing.