GSM3384170: Rec27-FLAG_interacting; Schizosaccharomyces pombe; ChIA-PET
Formaldehyde was added to a final concentration of 1% in 500 mL of culture. Chromatin was prepared and sonicated as in Fowler et al. 2013 Mol. Cell 49:983-996.  DNA was sheared to ~1000 bp and the epitope tagged protein of interest was immunoprecipitated.  For conformation capture samples ("interacting"), the immunoprecipitate was ligated while immobilized on magnetic beads and subsequently eluted.  Crosslinks were removed with high temperature incubation and protein digested using proteinase K.  For "de-crosslinked" samples, immunoprecipitated chromatin was eluted, crosslinks removed, and protein digested prior to DNA ligation.  In both cases, the ligated DNA was finally ethanol precipitated and amplified using one of two different kits (Sequenase Version 2.0 DNA Sequencing Kit, Affymetrix; or REPLI-g Single Cell Kit, Qiagen). DNA from all samples was amplified using either a Sequenase Version 2.0 DNA Sequencing Kit (for anti-GFP) or a Qiagen REPLI-g Single Cell Kit (for anti-FLAG).  DNA was quantified using a Bioanalyzer (Invitrogen).  Sequencing libraries were each prepared from 1 µg of DNA.  DNA was initially fragmented with a Covaris LE220 Focused-ultrasonicator (Covaris, Woburn, MA) using factory settings for an average size of 300 bp.  Library DNA was prepared using the KAPA DNA Library Preparation and HiFi PCR Kits (Kapa Biosystems, Wilmington, MA) on a PerkinElmer Sciclone NGSx Workstation (PerkinElmer, Waltham, MA) for Rec25-FLAG and Rec27-FLAG IPs, or the Epicentre Nextera DNA Sample Prep kit (Illumina, Inc., San Diego, CA) manually for Rec27-GFP IPs.
GEO
GSM3384170
GEO Accession
GSM3384170