GSM4105541: SOX4_IP2; Homo sapiens; ChIP-Seq
In brief, after HASOX4 overexpression, approximately total ~12×10e6 U251 cells (4 X 10cm plates) were crosslinked with methanol-free formaldehyde (Thermo Scientific, 28908). Then quench the crosslinking with glycine solution. After lysis and centrifugation, chromatin was sheared into 100-500 nucleotides length by Bioruptor Machine (Diagenode, B01010002). 10 µg chromatin solution was used for input. 150 µg of chromatin solution was applied for immunoprecipitation by 5 µg HA (Abcam; ab99110). Subsequently, 100 µl Magnetic Protein G Dynabeads (Life Technologies, 10003D) were resuspended in immunoprecipitation samples incubate for 2 hours at 4°C with gentle rotation to poll down primary antibody affinity chromatin fragments. After subsequently treated with RNase, Proteinase K and high concentration of NaCl solution (5M), Input and IP Chromatin was purified with a QIAquick PCR purification kit (Qiagen, 28104) according to kit manual. The chromatin concentration was determined by Qubit fluorometer and Qubit dsDNA HS assay kit (Life Technologies, Q32850) according to kit manual. For statistic, every groups were triplicated. Libraries were synthesized from 10 ng purified input chromatin and 10 ng purified immunoprecipitated chromatin using a NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina (New England Biolabs, E6240S) with NEBNext Multiplex Oligos for Illumina (New England Biolabs, E7335S). Replicate libraries were prepared from independent immunoprecipitations. ChIP-seq: single end 50-base length sequencing reads were generated on an Illumina HiSeq 2500 System.