title: GSM1533447: H3K4me3_ChIPSeq; Oreochromis niloticus; ChIP-Seq
library_construction_protocol: Tilapia fin tissue was dissected, immediately frozen on dry ice, and stored at _80¡C. ChIP-seq was performed based on Active Motif's HistonePath method (Active Motif) as previously described (Ramagopalan et al. 2010). The frozen tissue was crushed in liquid nitrogen and fixed in PBS + 1% formaldehyde at room temperature for 10 minutes. Fixation was stopped by the addition of 0.125 M glycine (final concentration), and the fin pieces were washed 2x in PBS and 1x in deoxycholate sonication buffer. Lysates were sonicated with a microtip in order to shear the DNA to an average length of 300-500 bp. Lysates were cleared by centrifugation and stored at -80¡C. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by phenol/chloroform extraction and ethanol precipitation. Purified DNA was quantified on a NanoDrop spectrophotometer. Extrapolation to the original chromatin volume allowed quantitation of the total chromatin yield. For each ChIP reaction, 15 µg of chromatin was pre-cleared with protein A agarose beads (Invitrogen). ChIP reactions were set up using pre-cleared chromatin and antibody H3K4me3 (Active Motif, cat. 39159, Lot# 4) and incubated overnight at 4¡C. Protein A agarose beads were added and incubation at 4¡C was continued for another 3 h. Immune complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase treatment and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65 ¡C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. ChIP DNA was prepared for amplification by converting overhangs into phosphorylated blunt ends and the addition of an adenine to the 3'-ends. Illumina genomic adapters were ligated and the sample was size-fractionated (200-300 bp) on an agarose gel. After a final PCR amplification step (18 cycles), the resulting DNA libraries were quantified and sequenced on Illumina MiSeq. Sequences (50 nt reads, single end).
attributes.tag: GEO Accession
attributes.value: GSM1533447