GSM4909192: Input RPO21-FLAG in DMSO; Saccharomyces cerevisiae; ChIP-Seq
Chromatin immunoprecipitation was performed as previously described, with the following details and modifications (Katou, Y., Kaneshiro, K., Aburatani, H. & Shirahige, K. Genomic approach for the understanding of dynamic aspect of chromosome behavior. Methods Enzymol 409, 389-410 (2006).). One hundred ml of cell culture (at an OD of 1.0) was crosslinked with 1 % formaldehyde for 30 minutes at room temperature, followed by incubation at 4°C overnight. Chromatin was sheared to a size of 300-500 base pairs by sonication (Bandelin Sonopuls HD 2070.2) and IP reactions, with anti-FLAG antibody (Sigma, F1804) conjugated to Dynabeads Protein A (Invitrogen, 10002D), were allowed to proceed overnight at 4°C. After completing the immunoprecipitation and reversing crosslinks as in 5, the DNA clean-up step was modified as follows. One hundred μl TE containing 10 μg RNase A (VWR, A3832) was added to IP and input fractions and the reactions were incubated for 1 hour at 37°C. Then, 2 μl of 50 mg ml-1 Proteinase K (Sigma, 000000003115879001) was added and the reactions were incubated for 2 hours at 37°C. Lastly, the DNA was purified using Qiagen PCR Purification kit according to standard instructions. DNA from ChIP and input fractions was further sheared to an average size of approximately 150 bp by sonication (Covaris M220). Samples were then prepared for sequencing using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645) according to the manufacture's protocol.