title: GSM3733685: Input [ChIP-Seq]; Danio rerio; ChIP-Seq
library_construction_protocol: Cells were lysed as described (Bogdanovic et al., 2013, Methods).  Chromatin was sonicated using a probe sonicator (SONOPULS, Bendelin) with a MS72 micro tip.  The sonication was performed with 20% amplitude with 12x3 cycles of 10 sec on, 50 sec off with 12 min on ice between each cycle.  After sonication, 120 μl of 10% Triton X-100 was added.  The size of sonicated chromatin was assessed by gel electrophoresis with fragment sizes between 100-300 bp.  ChIP was conducted as previously described (Bogdanovic et al., 2013, Methods), using Protein G Dynabeads (Invitrogen) and anti-HA antibody (Abcam, ab9110).  For the elution, we resuspended the beads in 100 μl of elution buffer (50mM Tris pH8.0, 10mM EDTA, and 1% SDS) at 65°C for 15 min and reversal of crosslinks was performed overnight at 65°C with shaking at 1000 rpm.  A total of 200 μl of TE buffer was added, and samples were incubated with RNaseA (final concentration 0.2 ug/mL) at 37°C for 2 h.  Then, proteinase K was added (final concentration 0.2 ug/mL) and samples were incubated at 55°C for 2h.  An equal amount (400 μl) of phenol:chloroform:isoamyl alcohol (25:24:1) was added and the aqueous phase extracted after centrifugation at 8,000 rpm for 5 min.  DNA was purified from the aqueous phase using the Qiagen MinElute PCR purification column according to manufacturer's protocol. After reverse cross-linking and cleanup samples were quantified by HS DNA Qubit measurement (Thermo) and 10ng DNA form Input and ChIP samples were used as input for TruSeq ChIP Sample Prep protocol following manufacturers protocol until ligation of adapters and cleanup. Ligated products were not separated by gel but used directly to PCR amplification followed by double sided bead cleanup (0.85x + 0.75x). Insert size and Library integrity was tested on LabChip Gx Touch 24 (Perkin Elmer) and quantified by Qubit HS DNA assay. Libraries were sequenced on Nextseq500 with V2 chemistry resulting in a minimum of 25M reads.
attributes.tag: GEO Accession
attributes.value: GSM3733685