GSM1217541: seq-SDQ5413_CEC7_fem2_AD_Input_Rep2; Caenorhabditis elegans; ChIP-Seq
Worm extract was made from adult worms.  Frozen worm popcorn was ground up in a mixer mill.  Samples were then fixed with 1% formaldehyde for 10 minutes. Chromatin was then sheared by sonication in the Bioruptor, and the soluble fraction was collected and stored at -80°C for future use. Worm_chromatin_immunoprecipitation_vCW2. ~100ug (~2ug DNA) extract was used for each ChIP with 5% 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 50 uL protein A or anti-IgG Dynabeads and washed 5 minutes with 1mL 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 75uL elution buffer for 15 minutes at 65C. Samples were treated with proteinase K at 55C for 1-2hrs then transferred to 65C overnight to reverse crosslinks. DNA was cleaned up with Ampure XP beads. For a detailed protocol see http://www.modencode.org/. Worm_ChIP-seq_ DNA_Library_Preparation_vSS2. ChIP and 10ng of 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.Changes: Ligate O/N at 16C. Use adapters and primer cocktail from Illumina TruSeq kit. Use AMPure cleanup in the place of Qiagne cleanup. Use Pippin prep for size selection . 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.
GEO Accession
GSM1217541