title: GSM4802308: HIF3a_input_1; Homo sapiens; ChIP-Seq
library_construction_protocol: The suspension was sonicated to generate DNA fragments of 200-500bp, centrifuged for 10 min at 12,000 g, and the supernatant was directly used for immunoprecipitation. For each immunoprecipitation, 1000μL cell extracts were incubated with HIF3A antibody (orb101652, Biobyt, China) overnight at 4 ℃. The immunoprecipitates were further incubated with protein A Dynabeads for 3h at 4 ℃. After applying to magnet and removing the supernatants, the beads were sequentially washed with lysis buffer, high-salt buffer (250 mM Tris 7.4, 750 mM NaCl, 10 mM EDTA, 0.1% SDS, 0.5% NP-40 and 0.5 deoxycholate), and PNK buffer (50 mM Tris, 20 mM EGTA and 0.5% NP-40) for two times, respectively. The immunoprecipitates were eluted from the beads with elution buffer (50 nM Tris 8.0, 10 mM EDTA and 1% SDS) and reverse cross-linked by overnight incubation at 65 ℃. After sequential RNase A and proteinase K treatment, DNA fragments were purified by phenol extraction and ethanol precipitation. To generate libraries for high-throughput sequencing, purified DNA fragments were end-repaired, adenylated, ligated to adaptors and PCR amplified for 12 cycles. The PCR products corresponding to 300-500 bps were gel purified, quantified and stored at -80 ℃ until used for sequencing. For high-throughput sequencing, the libraries were prepared following the manufacture's instructions and applied to Illumina X-Ten system with 150x2 paired-end type by Novogene.