title: GSM3592559: FOXL2_ChIPseq_Dox_WT_3; Homo sapiens; ChIP-Seq
library_construction_protocol: Crosslinked cells were quenched with one-tenth volume of 1.25 M Glycine (Sigma G7126) for 5 minutes and washed twice with dPBS before harvesting the cells. Cells were pelleted at 3,000 rpm, the supernatant removed and the cell pellet frozen at -80oC. Additionally, the control cell line, SVOG3e/rtTA/V5 that does not express V5-FOXL2, was seeded and treated as above for one time point at T=12 hours for  non- induced and Doxycycline-induced cells. Cell lysis, chromatin preparation, immunoprecipitation using anti-V5 antibody (Invitrogen, R960CUS) and recovery was performed as described previously (Pon et al. 2015), with the following modifications; the chromatin pellet was resuspended in 400 μl of ChIP IP buffer and sonicated (Duty Cycle 20%, Intensity 8,Cycles/burst 200, Time: 60 seconds) in a Covaris Cov-3 Sonicator. Pre-cleared chromatin (150 μg) was incubated with 4 μl (2 μg) of monoclonal mouse anti-V5 antibody (Invitrogen R960-25) for 1 hr at 4oC with rocking, followed by the addition of 40 μl of Protein G Dynabeads (Invitrogen) that was blocked with salmon sperm DNA and bovine serum albumin as previously described (3). Chromatin/antibody/Dynabead mixture was incubated overnight at 4oC on a nutator. The Dynabead bound proteins and associated chromatin was washed and eluted as previously described (3) and DNA was submitted for library construction and sequencing. ChIP-seq library construction was performed according to standard operating procedures available at http://www.epigenomes.ca/protocols-and-standards.
attributes.tag: GEO Accession
attributes.value: GSM3592559