title: GSM5253620: ChIP-seq_LSD1_OTUD7B KD; Homo sapiens; ChIP-Seq
library_construction_protocol: Briefly, cells were fixed with 1% formaldehyde (Sigma) for 10 min at room temperature (RT) (for H3Kme2 and H3K9me2 ChIP), or fixed with DSG (2 × 10−3 m) (Proteochem) for 45 min at RT (for LSD1 ChIP), and washed twice with PBS. Fixation was stopped by adding glycine (0.125 m) and incubated for 5 min at RT, followed by washing with PBS twice. Cells were then lysed using cell lysis buffer (50 × 10−3 m Tris‐HCl pH = 8.0, 0.5% NP40 and 50 × 10−3 m NaCl) to remove the cytosol proteins. The pellet was resuspended in 1% SDS lysis buffer (for H3K4me2 or H3K9me2 ChIP) or nuclear lysis buffer (1% Triton X‐100, 2 × 10−3 m EDTA pH = 8.0, 400 × 10−3 m NaCl, 20 × 10−3 m Tris‐HCl pH = 7.8, and 0.1% SDS) (for LSD1 ChIP). Chromatin DNA was sheared to 300–500 bp average in size through sonication. The lysates were immunoprecipitated with control IgG or the indicated specific antibodies overnight at 4 °C, followed by incubation with protein G magnetic beads (Invitrogen) for 4 h. After washing and elution, the protein–DNA complex was reversed by heating overnight at 65 °C. Immunoprecipitated DNA was purified by using QIAquick spin columns (Qiagen) and subjected to high‐throughput sequencing. The libraries were constructed following Illumina/NEB's Chip-Seq Sample prep kit. Briefly, Chip DNA was end-blunted and added with an 'A' base so the adaptors from Illumina with a 'T' can ligate on the ends. Then 200–400 bp fragments are gel-isolated and purified. The library was amplified by 18 cycles of PCR.