title: GSM9003236: CHIPseq-TY, WT, rep1; Homo sapiens; ChIP-Seq
library_construction_protocol: Attached cells were crosslinked with 0.5 mg/mL disuccinimidyl glutarate (DSG; Thermo Fisher) in PBS for 45 min at room temperature. Cells were washed once with PBS followed by incubation with 1% FA for 20 min at RT. Fixation was stopped by the addition of glycine in PBS to a final concentration of 0.1 M for 3 min at RT. This was followed by washing with cold PBS and collection of the cells in 0.25% Triton X-100, 10 mM EDTA (pH 8.0), 0.5 mM EGTA (pH 8.0), and 20 mM Hepes (pH 7.6) in miliQ. Chromatin was pelleted by centrifugation for 5 min at 400 × g and incubated in 150 mM NaCl, 1 mM EDTA (pH 8.0), 0.5 mM EGTA (pH 8.0) and 50 mM Hepes (pH 7.6) in miliQ for 10 min at 4 °C. Chromatin was again pelleted by centrifugation and resuspended in ChIP-buffer (0.15 % SDS, 1 % Triton X-100, 150 mM NaCl, 1 mM EDTA (pH 8.0), 0.5 mM EGTA (pH 8.0) and 20 mM Hepes (pH 7.6) in miliQ) to a final concentration of 15 × 10^6 cells/mL. Chromatin was sonicated to approximately one nucleosome using the Bioruptor Pico (Diagenode), 8 cycles of 30s ON/30 s OFF in a 4°C water bath. ChIP was performed using 84 μg of WT chromatin and 60 ug of CFAP20-KO + TY-CFAP20 chromatin + 74 and 53 ng of Drosophila spike-in chromatin, respectively (Active Motif, 53083) with 5.7 ug of TY antibody (Diagenode; C15200054) and 1 μg spike-in antibody (Active Motif, 61686) by overnight incubation at 4 °C. Protein-chromatin-pull down followed with 20 ul protein A Dynabeads (Thermo Fisher; 10001D) per sample. ChIP samples were extensively washed and purified using Qiagen MinElute kit. Sample libraries were prepared using Hifi Kapa sample prep kit and A-T mediated ligation of Nextflex adapters or xGen UDI-UMI adapters. Samples were sequenced using an Illumina NovaSeq X, using paired-end sequencing with 151 bp from each end. ChIP-seq