title: GSM3184717: ChIP-seq of  H3K4me3_siRAD21_E2_MCF7; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were cross-linked with 1% formaldehyde at room temperature for 10 min. Or for some cases, cells were double cross-linked with 1mM DSG (ProteoChem) for 1 hr first and then for 10minutes by 1% formaldehyde. In both situations, the crosslinking was quenched by addition of 0.125M glycine for 10 minutes. ChIP chromatin was fragmented using (Diagenode) Bioruptor®300 (20-40 cycles, 30sec on, 30sec off, 4°C). Subsequently, the soluble chromatin was harvested by 16,100g centrifugation, pre-cleared with 10-20ul Dynabeads G (Life Technologies), and was then incubated with 1-5 μg of antibodies at 4°C overnight. The next morning, immuno-precipitated complexes were collected using 30ul Dynabeads protein G (Invitrogen) per reaction. The immune-complexes were subjected to washes with wash buffer I for once, with wash buffer II for twice, and with Tris-EDTA (TE) + 0.1% triton x-100 for once, with TE for once, the beads were incubated at 55°C for 2hrs with proteinase K and de-crosslinked at 65°C for overnight. The final ChIP DNA was extracted and purified using QIAquick spin columns (Qiagen). The library was processed that the extracted DNA was ligated to specific adaptors for Illumina's HiSeq system using KAPA Hyper Prep Kit (Kapa Biosystems)