alias: GSM1195432
title: GSM1195432: seq-AB46540_NIgG_N2_Mxemb_Input_Rep2; Caenorhabditis elegans; ChIP-Seq
library_construction_protocol: Worm_embryo_extraction_vSE3. Embryos were resuspended in FA buffer (50 mM HEPES/KOH pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1 % sodium deoxycholate; 150 mM NaCl) + protease inhibitors (Calbiochem Cat# 539131) + phosphatase inhibitors (Calbiochem Cat# 524624), and dounced 30 times. Using samples were sonicated in Bioruptor using settings: high 30 sec on 30 sec off 15 minutes. Cell debris was removed by centrifuging at 13,000 g for 15 minutes at 4ºC, and taking the supernatant for ChIP. For a detailed protocol see http://www.modencode.org/. Worm_chromatin_immunoprecipitation_v3. 2mg extract was used for each ChIP with 10% taken as input directly into elution buffer (1% SDS in TE, 250 mM NaCl) and treated with RNAse. Antibody was added to each IP sample and incubated overnight at 4C. Immune complexes were incubated (2hrs at 4C) with 20 uL protein A sepharose (Amersham) and washed 5 minutes with 1.5mL of each of the following solutions: ChIP Buffer, ChIP Buffer+1M NaCl, ChIP Buffer+500mM NaCl, TEL Buffer (10mM Tris-HCl pH 8.0, 250mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1mM EDTA), and 2X TE (10mM Tris-HCl pH 8.0, 1mM EDTA). Samples were eluted twice with 150uL elution buffer for 15minutes at 65C. Samples were treated with proteinase K at 55C for 1-2hrs then transfer to 65C overnight to reverse crosslinks. DNA was cleaned up Qiagen PCR purification kit and concentrated using by Speed vac. ChIP and input are blunted (T4 polymerase), an A-overhang is added (Klenow), DNA is ligated (concentrated T4 ligase) to annealed adaptors, size-selected, and amplified by PCR. Prepared sample are sequenced using Illumina GAII or HiSeq2000 at the High Throughput Sequencing Facility of University of North Carolina at Chapel Hill or Cambridge.
identifiers.namespace: GEO
identifiers.id: GSM1195432
attributes.tag: GEO Accession
attributes.value: GSM1195432