GSM8573251: BEAS-2B with Nrf2 knockout, 1 μM As³⁺ for 6 hours, KLF4, ChIP; Homo sapiens; ChIP-Seq
Cells were fixed for 15 min at 25 °C with 4% formaldehyde, and the reaction was halted by adding 125 mM glycine for 5 min at room temperature. Fixed cells were washed with PBS, scraped from the culture surface, and chromatin was isolated using lysis buffer and disrupted with a Dounce homogenizer. The lysates were sonicated to shear DNA to an average length of 300–500 bp. Genomic DNA (Input) was prepared by treating chromatin with RNase and proteinase K, followed by heating for de-crosslinking and ethanol precipitation. The DNA was quantified using a NanoDrop spectrophotometer (Thermo Scientific) and the total chromatin yield was extrapolated. A 30 μg aliquot of chromatin was precleared with protein A agarose beads (Invitrogen). ChIP was performed using 4 μg of antibodies against H3K4me1 (Thermofisher, cat#710795), H3K4me3 (Thermofisher, cat# 711958), H3K9me3 (CST, cat#4658), H3K27ac (CST, cat#8173), and KLF4 (R&D Systems, cat#AF3640). Complexes were washed, eluted with SDS buffer, treated with RNase and proteinase K, and crosslinks were reversed by overnight incubation at 65°C. ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. Illumina sequencing libraries were prepared through end-polishing, dA-addition, and adapter ligation, followed by PCR amplification. Sequencing was performed using Illumina NextSeq 500, and 75-nt reads were aligned to the GRCh38/hg38 genome using the Burrows-Wheeler Aligner (BWA) with default settings.