title: GSM8715085: Input, SETD6 KO, Rep1; Homo sapiens; ChIP-Seq
library_construction_protocol: For the DU145 CRISPR/Cas9 generated SETD6 knock-out, single clones were confirmed by sequencing as described in previously (Kublanovsky et al., 2023) Approximately 2 × 107 cells were washed with 1X PBS buffer (Gibco) and harvested by centrifugation at 500 g for 5 min. Next, formaldehyde was added directly to the cell culture media to a final concentration of 2%. After 10 min of incubation at room temperature, glycine was added to a concentration of 125 mM to quench crosslinking reaction. After that, the Magna ChIP™ HiSens Chromatin Immunoprecipitation Kit (Milipore, MAGNA0025) was used to remove cytosolic proteins. To digest chromatin fraction, the nuclear pellet was sonicated with EpiShear™ Probe Sonicator (Active Motif) at 4°C. In the following, ChIP preparation steps were processed according to the manufacturer's instructions. For each ChIP, 10 μg Flag antibody (Invitrogen, F1804) was used for enrichment of sample. All library preparation steps were conducted according to manufacturer's instructions of NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 1) (NEB). The ChIP-seq library was send to Novogene (UK) for Next-Generation-Sequencing (Illumina Novaseq 6000 system, paired-end, 2× 150 bp-mode).