GSM8745530: MCF-7 1456 KD-2 Rep1, PHF8 ChIP; Homo sapiens; ChIP-Seq
Cells were cross-linked with 1% formaldehyde in PBS for 10 minutes at 37°C, quenched by the addition of 125 mM glycine, and incubated 5 minutes at 4°C. The cross-linked cells were collected in ice-cold PBS and pelleted by centrifugation. The cells were then resuspended by gentle mixing by pipetting in ice-cold Farnham Lysis Buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40) with freshly added 1 mM DTT and 1x protease inhibitor cocktail. The supernatant was removed, and the crude nuclear pellet was collected by centrifugation and resuspended in SDS Lysis Buffer (50 mM Tris-HCl pH 7.9, 1% SDS, 10 mM EDTA) with freshly added 1 mM DTT and 1x protease inhibitor cocktail. After a 10-minute incubation on ice, the lysate was sheared by sonication using a Bioruptor (Diagenode) to generate chromatin fragments of approximately 250 bp in length. The sheared chromatin was clarified by centrifugation and the diluted ten-fold in ChIP Dilution Buffer (20 mM Tris-HCl pH 7.9, 0.5% Triton X-100, 2 mM EDTA, 150 mM NaCl) with freshly added 1 mM DTT and 1x protease inhibitor cocktail. ChIP libraries were prepared using a modified KAPA LTP Library Preparation kit (KAPA BiosystemsKK8232) for Illumina Platforms. Ten ng of sheared DNA was used to repair the ends of the damaged fragments using a proprietary master mix. The resulted blunted fragments were 3′ A-tailed using a proprietary mixture of enzymes to allow ligation to the specific Illumina adaptors. Each of the steps (i.e., end repair, 3'A tailing, and adaptor ligation) was followed by AMPure XP bead clean up (Beckman Coulter, A63881). After adapter ligation, DNA enrichment was performed using Kapa HiFi Hot Start Ready PCR mix, and a cocktail of primers (1 cycle at 98 °C for 45 seconds; 5 cycles at 98 °C for 20 seconds, 63 °C for 30 seconds, and 72 °C for 30 seconds; and 1 cycle at 72 °C for 1 min), and purified with AMPure XP beads. DNA templates were size selected (~200-300 bp) by running on a 2% agarose gel, followed by PCR enrichment (1 cycle at 98 °C for 45 seconds; 11 cycles at 98 °C for 20 seconds, 63 °C for 30 seconds, and 72 °C for 30 seconds; and 1 cycle at 72 °C for 1 min) and final purification. The quality of the final libraries was assessed using a 2200 TapeStation (Agilent Technologies). The libraries were quantified using Qubit dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific, Q32854) and samples pooled at final concentration of 2 nM. Library sequencing. The libraries were sequenced using a NextSeq sequencer (Illumina; Single-end reads, 75 bp for all samples). At least two biological replicates were sequenced for each cell line for a minimum of roughly 100 million raw reads per cell line.