Worm_germlineless_adult_soma_extract_v2.2. Frozen worms were ground to a powder using mortar and pestle. Worm powder was diluted in M9 + 1% formaldehyde+1mM PMSF and fixed 5 or 10 minutes. Fixing was quenched by the addition of 125 mM glycine for 5 minutes. Cells were collected by centrifuging 5 minutes at 4000 g at 4C. Samples were washed and 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). Using a Branson sonifier microtip, samples were sonicated on ice at the following settings: 35% amplitude, 0.9 sec on, 0.1 sec off, 12 pulses, 10 times. Cell debris was removed by centrifuging at 13,000 g for 15 minutes at 4?C and taking the supernatant. Protein concentration was determined by Bradford Assay and 10 mgs were used per ChIP. For a detailed protocol see http://www.modencode.org/.
Growth protocol
Worm_L4_growth_and_harvest_vSE1. Worms were grown in standard S-basal media liquid culture. The culture was synchronized by starving hatched embryos at L1 stage and grown at 20C in liquid culture until they reach L4 stage. Worms were collected over 34 uM Nitex membrane, and washed with M9 Buffer + protease inhibitors (Calbiochem). Worms were resuspended in 1 pellet volume of M9, frozen as popcorn in liquid N2, and stored at -80C. For a detailed protocol see http://www.modencode.org/.
Extracted molecule
genomic DNA
Extraction protocol
Worm_chromatin_immunoprecipitation_v4.1. 2.2 mg extract was taken and sarkosyl was added to 1% final concentration. 0.2 mg 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 mL 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. For a detailed protocol see http://www.modencode.org/.Samples were amplified using a modified Whole Genome Amplification Kit protocol (Sigma).
Worm_germlineless_adult_soma_extract_v2.2. Frozen worms were ground to a powder using mortar and pestle. Worm powder was diluted in M9 + 1% formaldehyde+1mM PMSF and fixed 5 or 10 minutes. Fixing was quenched by the addition of 125 mM glycine for 5 minutes. Cells were collected by centrifuging 5 minutes at 4000 g at 4C. Samples were washed and 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). Using a Branson sonifier microtip, samples were sonicated on ice at the following settings: 35% amplitude, 0.9 sec on, 0.1 sec off, 12 pulses, 10 times. Cell debris was removed by centrifuging at 13,000 g for 15 minutes at 4?C and taking the supernatant. Protein concentration was determined by Bradford Assay and 10 mgs were used per ChIP. For a detailed protocol see http://www.modencode.org/.
Growth protocol
Worm_L4_growth_and_harvest_vSE1. Worms were grown in standard S-basal media liquid culture. The culture was synchronized by starving hatched embryos at L1 stage and grown at 20C in liquid culture until they reach L4 stage. Worms were collected over 34 uM Nitex membrane, and washed with M9 Buffer + protease inhibitors (Calbiochem). Worms were resuspended in 1 pellet volume of M9, frozen as popcorn in liquid N2, and stored at -80C. For a detailed protocol see http://www.modencode.org/.
Extracted molecule
genomic DNA
Extraction protocol
Worm_chromatin_immunoprecipitation_v4.1. 2.2 mg extract was taken and sarkosyl was added to 1% final concentration. 0.2 mg 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 mL 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. For a detailed protocol see http://www.modencode.org/.Samples were amplified using a modified Whole Genome Amplification Kit protocol (Sigma).
ChIP-chip_label_hyb_nimblegen_v1. DNA was labeled and hybridized to C. elegans tiling array by Roche NimbleGen according to the protocol described in chapter 3 and 4 of the NimbleGen Arrays User?s Guide ChIP-chip Analysis, Version 3.1, 27 May 2008. Briefly, Amplified IP or input DNA was either labeled with Cy5 or Cy3 in the presence of Klenow fragment. The reaction was stopped by the addition of EDTA. Labeled DNA was recovered by isopropanol precipitation, and dried. The labeled DNA was hybridized to C. elegans tiling array for 16 - 20 hours at 42?C.
Scan protocol
ChIP-chip_scanning_nimblegen_v1. Array scanning and raw data extraction were performed at Roche NimbleGen, according to the protocol described in chapter 5 and 6 of the NimbleGen Arrays User?s Guide ChIP-chip Analysis, Version 3.1, 27 May 2008. Briefly, array signal was scanned by using a GenePix 4000B Scanner with associated software and saved as .tif files of the 532nm and 635nm images individually. Raw signal intensities of the images were extracted and saved as .pair files by using NimbleScan software according to the NimbleScan v2.4 User?s Guide.
Description
PolII ChIP data in N2 L4s, replicate 1, array part 1. A mouse monoclonal antibody which recognizes primarily the unphosphorylated RNA Pol II CTD (8WG16) obtained from Covance (MMS126R) was used for ChIP.
Data processing
ChIP-chip_normalization_standard_zscore_v2. First, the ratio of intensity from sample/reference channel was transformed to log2 space. Second, z-scores were obtained for each probe by standardizing the data by subtracting the mean and dividing by the standard deviation of all probes.
