title: GSM2563172: 288_Fish_D_C5_120_H3k27ac_GGCTAC_L001_R1_001; Gasterosteus aculeatus; ChIP-Seq
library_construction_protocol: For tissue intended for ChIP-Seq, brain tissue from regions of interest was pooled and kept at 4° C until homogenization in PBS with protease inhibitor cocktail (PIC, Roche, Basel, Switzerland). Fresh brains were homogenized in pools of five on ice by motorized pestle. Homogenized brains were fixed in PBS with 1% formaldehyde for 10 minutes. Fixing reaction was stopped with addition of Glycine to 0.125M. Fixed cells were washed 3x with PBS+PIC to remove formaldehyde. Washed cells were lysed to nuclei with lysis solution – 50 mM Tris-HCl (pH 8.0), 2 mM EDTA, 0.1% v/v NP-40, 10% v/v glycerol, and PIC – for 30 minutes on ice. Cell debris was washed away with PBS with PIC. Nuclei were pelleted and flash-frozen on dry ice.  Frozen nuclei were thawed on ice in lysis solution (1% SDS, 10mM EDTA, 50mM Tris, pH 8.1 with protease inhibitor cocktail). Nuclei were counted using a hemocytometer. Nuclei were sonicated at high power for 7 x 7 minute cycles (30 s on, 30 s off) in a BiorupterTM UCD-200 (Diagenode, Liège, Belgium) sonicator. Remaining cellular debris was pelleted by centrifugation for 10 minutes at 13,000 x g. Fragmented chromatin was processed for histone H3K27Ac ChIP (Abcam ab4729) with Diagenode iDeal ChIP kits (Diagenode #C01010051, Liège, Belgium), according to manufacturer’s specifications with minor adjustments. One million nuclei were used for each IP. 25 µl of each IP was reserved for input samples. Technical replicate inputs were pooled to 50 µl. 2 ug of h3k27ac antibody was used for each IP. An additional wash in TE buffer was performed after the initial four IP washes. Chromatin was processed for ESRRA ChIP using a customized protocol. Briefly, sonicated samples were pre-incubated with Protein G Dynabeads (Invitrogen 10009D) for three hours. Beads were removed and 10 µg of ESRRA antibody (SC-66882, Santa Cruz Biotechnology, Dallas, TX, USA) was added overnight with rotation at 4° C. Protein G beads were added for 3 hours to bind antibodies. Beads were washed with high salt, low salt, LiCl, and TE buffers for 5 minutes each in succession. Precipitated chromatin was eluted in ChIP elution buffer (1% SDS, 0.1 M NaHCO3) for 15 minutes twice in 25 µl. Samples were reverse cross-linked at 65° C with 1,300 rpm rotation overnight. Samples were phenol-chloroform extracted with standard methods and eluted into 20 µl of nuclease-free water.  After ChIP, immunoprecipitated DNA was quantified using a Qubit 2.0 (Life Technologies, Carlsbad, CA, USA) with a dsDNA HS Assay kit (Life Technologies #Q32854). Libraries were prepared using KAPA LTP library kits (KK8230), with protocol as written, using Bioo Scientific index adapters. Libraries were size selected using AmpureXP beads (Beckman Coulter, Brea, CA, USA), with protocol as written, selecting for DNA between 200-500bp in size. Library quality was checked by Qubit 2.0 and Bioanalyzer (Agilent 2100). Samples were sequenced with an Illumina HiSeq 2500 sequencer using a TruSeq SBS sequencing kit, version 4. All samples were sequenced in single end format with fragment length of 100 bp.
attributes.tag: GEO Accession
attributes.value: GSM2563172