Embryos were prepared by bleaching from gravid N2 adults grown in S-basal media liquid culture. Live embryos were cross-linked using 2% formaldehyde for 30 minutes at room temperature followed by quenching with 125mM glycine for 15 minutes. Embryos were then washed twice with M9 Buffer and once by ChIP buffer. Pellets were frozen at -80C.
Growth protocol
Standard C. elegans growth in liquid culture for N2 wildtype.
Extracted molecule
genomic DNA
Extraction protocol
Extracts were prepared by resuspending embryo pellets in 1 volume ChIP Buffer (50mM HEPES-KOH pH 7.5, 300mM NaCl, 1mM EDTA pH 8.0, 1% TritonX-100, 0.1% sodium deoxycholate, 10% glycerol, protease inhibitors (Calbiochem)), followed by dounce homogenization (50X) and sonication (4X, 1s on, 0.5s off, at 20% amplitude on ice) using a Branson Sonifier. In a volume of 500uL, 2mg extract was used for each ChIP with 5% taken as Input directly into 400uL elution buffer (0.1M NaHCO3, 1% SDS). 2-6ug antibody was added to each IP sample and incubated overnight at 4C. Immune complexes were pulled down with 10uL protein-A or protein G sepharose (Amersham) and washed 5 minutes with 1.5mL of each of the following solutions: ChIP Buffer, ChIP Buffer with 500mM or 1M NaCl, LiCl solution (10mM Tris-HCl pH 8.0, 250mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1mM EDTA), and TE (10mM Tris-HCl pH 8.0, 1mM EDTA). Samples were treated with 20ug RNAse for 30 minutes at 37C. IP samples were eluted twice with 200uL elution buffer. 16ul NaCl (200mM final) was added to each sample and crosslinks were reversed by incubation overnight at 65C. DNA was cleaned up using Zymo (Zymo Research) DNA purification columns.
Label
Cy5
Label protocol
Samples were amplified and labeled using LM-PCR as described in Ercan et al. (2007) Nat Genet. 2007 Mar;39(3):403-8.
Channel 2
Source name
Input gDNA from ChIP in N2 embryos, labeled with Cy3
Embryos were prepared by bleaching from gravid N2 adults grown in S-basal media liquid culture. Live embryos were cross-linked using 2% formaldehyde for 30 minutes at room temperature followed by quenching with 125mM glycine for 15 minutes. Embryos were then washed twice with M9 Buffer and once by ChIP buffer. Pellets were frozen at -80C.
Growth protocol
Standard C. elegans growth in liquid culture for N2 wildtype.
Extracted molecule
genomic DNA
Extraction protocol
Extracts were prepared by resuspending embryo pellets in 1 volume ChIP Buffer (50mM HEPES-KOH pH 7.5, 300mM NaCl, 1mM EDTA pH 8.0, 1% TritonX-100, 0.1% sodium deoxycholate, 10% glycerol, protease inhibitors (Calbiochem)), followed by dounce homogenization (50X) and sonication (4X, 1s on, 0.5s off, at 20% amplitude on ice) using a Branson Sonifier. In a volume of 500uL, 2mg extract was used for each ChIP with 5% taken as Input directly into 400uL elution buffer (0.1M NaHCO3, 1% SDS). 2-6ug antibody was added to each IP sample and incubated overnight at 4C. Immune complexes were pulled down with 10uL protein-A or protein G sepharose (Amersham) and washed 5 minutes with 1.5mL of each of the following solutions: ChIP Buffer, ChIP Buffer with 500mM or 1M NaCl, LiCl solution (10mM Tris-HCl pH 8.0, 250mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1mM EDTA), and TE (10mM Tris-HCl pH 8.0, 1mM EDTA). Samples were treated with 20ug RNAse for 30 minutes at 37C. IP samples were eluted twice with 200uL elution buffer. 16ul NaCl (200mM final) was added to each sample and crosslinks were reversed by incubation overnight at 65C. DNA was cleaned up using Zymo (Zymo Research) DNA purification columns.
Label
Cy3
Label protocol
Samples were amplified and labeled using LM-PCR as described in Ercan et al. (2007) Nat Genet. 2007 Mar;39(3):403-8. PMID: 17293863
Hybridization protocol
Samples were hybridized by Nimblegen Systems, Inc. Singh-Gasson S, Green RD, Yue Y, Nelson C, Blattner F, Sussman MR, Cerrina F. Maskless fabrication of light-directed oligonucleotide microarrays using a digital micromirror array. Nat Biotechnol. 1999 Oct;17(10):974-8. PMID: 10504697
Scan protocol
Samples were scanned by Nimblegen Systems, Inc. Singh-Gasson S, Green RD, Yue Y, Nelson C, Blattner F, Sussman MR, Cerrina F. Maskless fabrication of light-directed oligonucleotide microarrays using a digital micromirror array. Nat Biotechnol. 1999 Oct;17(10):974-8. PMID: 10504697
Description
NA
Data processing
Data was normalized by median centering log2 ratios (IP/input). Log ratios from each experiment were converted to z-scores.