GSM1520924: Lib152; Homo sapiens; ChIP-Seq
For ChIP assays, normally, cells were fixed with 0.4% (v/v) formaldehyde at room temperature for 10 min. For improving the ChIP efficiency of non-DNA binding factors, double fixation was used. For double fixation with EGS (Pierce, Cat. no. 21565) and formaldehyde, cells were fixed initially with 1.5 mM EGS at room temperature for 30 min, and subsequently with 0.4% formaldehyde at room temperature for 10 min. For double fixation with DMA (Pierce, Cat. no. 20660) and formaldehyde, cells were fixed initially with 25 mM DMA at room temperature for 1 hour, and subsequently with 0.4% formaldehyde at room temperature for 10 min. After two washes with PBS, fixed cells were resuspended in ice-cold RIPA-0.3 buffer (10 mM Tris-HCl, 1mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% NaDOC, and 0.3 M NaCl, pH7.4) at the concentration of 40 million/ml, and chromatin was disrupted by sonication to the size range of 100 to 500 bp. Antibodies were diluted in RIPA-0.3, and bound to Dynabeads protein A (Life Technologies, cat. no. 10002D) by incubating at 4 °C for 3 hours. The bead-antibody complexes were washed with RIPA-0.3 twice, and then incubated with sonicated chromatin at 4 °C overnight. The second day, after 2 washes with RIPA-0.5, 1 wash with RIPA-0.3, 1 wash with RIPA-0, 2 washes with LiCl buffer (10 mM Tris-HCl, 0.25 M LiCl, 0.25% NP-40, and 0,25% NaDOC, pH7.4), and 2 washes with TE buffer, bound protein-DNA complexes were resuspended in elution buffer (10 mM Tris-HCl, 1mM EDTA, and 1% SDS, pH7.4) supplemented with 10 µg/ml RNase A for elution and RNA digestion, and incubated at 55 °C for 1 hour. Afterwards, proteinase K was added to the final concentration of 100 µg/ml, and 30 min later, the temperature of incubation was increased to 65 °C for decrosslinking.  After decrosslinking for 4–6 hours, DNA was purified by ChIP DNA Clean & Concentrator (Zymo Research, cat. no. D5205). RNA was extracted from cells using RNeasy Plus Mini Kit (Qiagen, cat. no. 74134) or Quick-RNA MiniPrep Kit (Zymo, R1054) by following the manufacturer’s protocol (Qiagen, cat. no. 74134). ChIP-seq libraries were constructed with 5 to 10 ng immunoprecipitated DNA. RNA-seq libraries were prepared by following a previously published strand-specific protocol. Samples were sequenced on HiSeq 2000 or 2500 by following the manufacturer's protocols.