GSM3187964
GSM3187964: Early embryo input, rep2; Caenorhabditis elegans; ChIP-Seq
Embryos were resuspended in FA buffer (50 mM HEPES/KOH pH 7.5, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate; 150 mM NaCl) + protease inhibitors. Sample was dounced 30 times using tight pestle at 4ºC, then sonicated 9 times on ice at the following settings: 30 sec; power output 3; 70% duty cycle. Cell debris was removed by centrifuging at 13,000 rpm for 15 minutes at 4ºC. The supernatant was filtered through a Millipore Ultrafree-MC 0.45 um filter unit (cat. UFC30HV0S) at 13,000 rpm, 4°C for 1 minute to remove lipids. Protein concentration was determined, and the extract was aliquoted and stored at -80ºC. For a detailed protocol see http://www.modencode.org/. DNA was incubated with an End Repair Enzyme mix (NEB Klenow, T4 DNA polymerase and T4 PNK) to ensure blunt ends, then purified and incubated with Exo(-) Klenow fragment in the presence of dATP to add adenosine at the 3' ends (a single A overhang for more efficient and directed ligation of the adaptors). After a second purification, the DNA fragments were ligated with single-end 'Homebrew' adaptors that contained an index sequence within. After ligation, the samples were purified twice using SPRI beads, allowing for a size selection step to get rid of excess adaptors. Samples were then amplified by PCR with single-end primers. Depending on the original fragmentation of DNA, samples were purified by SPRI or by separation and purification from an agarose gel.
GEO
GSM3187964
GEO Accession
GSM3187964