title: GSM1259391: EAP1-null; Caenorhabditis elegans; ChIP-Seq
library_construction_protocol: Chromatin preparation was performed as described in (Stock et al., 2007) with some modifications. Strains were maintained at 16°C until the appropriate generation and then shifted to 25°C after birth. 50 µl packed young adult worms were subjected to five freeze/thaw cycles in liquid N2, and fixed in 1% formaldehyde in PBS (10 min, RT). During fixation, worms were dounced 100 times in a glass homogenizer. Fixation was stopped by addition of Glycine to 0.125M (5 min, RT). Worms were washed in cold PBS, before “swelling” buffer (25 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl and 0.1% NP-40) was added to lyse the cells (10 min, 4°C). After resuspension in “sonication” buffer (50 mM HEPES pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate and 0.1% SDS), nuclei were sonicated using a Misonix 3000 (Amplitude 60; 25 cycles; 15s ‘on’, 45s ‘off’; 4°C). The resulting material was centrifuged twice (10 min, 4°C) at 14,000 rpm. Swelling and sonication buffers were supplemented with 5 mM NaF, 1 mM PMSF, and protease inhibitor cocktail (Roche). ChIP-seq libraries were prepared using NEBNext DNA library preparation reagents (E6000) and the protocol and reagents concentrations described in the Illumina Multiplex ChIP-seq DNA sample Prep Kit. Libraries were indexed using a single indexed PCR primer. After PCR amplification, 300-600 bp DNA fragments were selected on an agarose gel. Libraries were quantified by Qubit (Invitrogen), and library size was assessed by Bioanalyzer (Agilent). Libraries were sequenced using a HiSeq 2000 (Illumina) to generate 50 bp single end reads. ChIP-seq libraries were generated from two biological repeats for G0 and G20, a single repeat for G10, and four Input samples.
attributes.tag: GEO Accession
attributes.value: GSM1259391