title: GSM9703679: Mel270 HM3#11 Input; Homo sapiens; ChIP-Seq
library_construction_protocol: RNA-seq：1) For THZ1 treatment, Mel270 cells were incubated in medium containing THZ1 at the indicated.concentrations or DMSO for 6 h, RNA was isolated with Rneasy Micro Kit (Qiagen). 2) For exosomal RNA-Seq, RNA extraceted from HUVEC exosomes was treated with terminator 5'-Phosphate-Dependent Exonuclease (Epicenter, Madsion, WI) to degrade 5'-monophosphate RNA in exosomal RNA, and subsequently treated with 5' polyphosphatase (Epicenter, Madsion, WI) to convert 5'ppp to 5'p, yielding specifically ligated RNA adaptor P5_RNA to RNAs that originally have 5'ppp. ChIP-seq：EZ-ChIP Kit (EMD Millipore, Burlington, MA) was used for ChIP according to our previous reports⁹. In brief, Mel270 cells (1×10⁷) were crosslinked in 1% formaldehyde for 10 min at room temperature and quenched unreacted formaldehyde with glycine. The cell lysates were sonicated to shear DNA to length of 200–500 bp and centrifuged at 12,000 g at 4 °C for 10 min. Protein G agarose (60 μL) was added to each tube to preclear the chromatin before immunoprecipitated with anti-H3K27ac antibodies (5 μg), anti-ELK-1 antibodies (5 μg), anti-STAT1 antibodies (5 μg) or rabbit IgG (negative Parental) overnight at 4 °C with rotation. The protein-DNA complexes underwent reverse crosslinks overnight. RNase A and Proteinase K were used to digest RNA and protein, respectively. DNA was then purified with spin columns. ATAC-seq: CFPAC-1 (pTSB-Vector or pTSB-KLF4; Scramble, shKLF4#1 or shKLF4#2) cells were digested with 0.25% Trypsin. After washing with PBS, 5 × 10^4 cells were lysed in ice-cold lysis buffer (10 mmol/L Tris-HCl pH 7.5, 10 mmol/L NaCl, 3 mmol/L MgCl2, 0.5% IGEPAL CA-630, and 0.1% Tween-20) for 10 min. After nuclei were collected by centrifuging at 500 g for 5 min, Tn5 transposase reactions (#TD501, Vazyme Biotech, Nanjing, China) were immediately performed at 37°C for 30 min. The transposed DNA fragments were purified with VAHTS DNA Clean Beads (#N411, Vazyme Biotech, Nanjing, China). RNA-seq：Sequencing libraries were generated using NEBNext® UltraTMRNA Library Prep Kit.for Illumina® (New England Biolabs, Ipswich, MA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. ChIP-seq： Illumina sequencing libraries were generated using TruSeq PE Cluster Kit v3-cBot-HS and sequenced on an Illumina HiSeq platform. ATAC-seq：The purified DNA fragments were amplified using TruePrep Index Kit V2 for Illumina (#TD202, Vazyme Biotech, Nanjing, China) according to the manufacturer's instructions.