GSM1217528: seq-302-32369_H3K9me2_ojIs9_YA_germline_ChIP_Rep2; Caenorhabditis elegans; ChIP-Seq
Worm_adult_purified_germline_extract_v1. Frozen worms were ground to a powder on liquid nitrogen using a Retsch mixer mill. Worm powder was dissolved into 10V NIBS buffer (25 mM HEPES pH 7.4, 118 mM NaCl, 48 mM KCl, 250 mM sucrose, 5% glycerol, 5 mM MgCl2, 0.5 mM spermine, 0.2 mM spermidine, 0.2 mM DTT, 1X protease inhibitor cocktail) and pelleted at 4000 rpm for 10 minutes at 4C. The pellet was resuspended in 2V NIBS and homogenized 10X with Potter-Elvehjem pestle. The nuclei suspension was then centrifuged once at 50 x g for 5 minutes at 4C and the supernatant was transferred to a fresh chilled tube. The nuclei suspension was then centrifuged once at 100 x g for 5 minutes at 4C and the supernatant was carefully transferred to a fresh chilled tube. This nuclei suspension was combined with prepared GFP binding nanobody-coated beads and allowed to incubate at 4C for 30 minutes. Bead nuclei complexes were then washed 2X with NIBS and fixed with NIBS + 1% formaldehyde for 5 minutes. Fixing was quenched by washing with NIBS + 125mM glycine, followed by NIBS only. Nuclei were resuspended in FA lysis buffer to lyse and sonicated using a Bioruptor XL at maximum intensity for 45 minutes (30 s on, 1 min off). The extract was then centrifuged at 15000 rpm / 15 minutes / 4C. Supernatants were pooled and assayed for quality by preparing DNA, quantifying protein by Bradford assay, and assaying germline-specific protein enrichment by western. Input and purified nuclei slide were examined for nuclei quality and enrichkment. Supernatants were aliquoted and stored at -80C. For a detailed protocol see http://www.modencode.org/. 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
GSM1217528