genotype/variation: wild type N2 gender: XX developmental stage: larval stage L1 sample type: input
Treatment protocol
Briefly, gravid hermaphrodites were bleached and embryos were synchronized in M9 for 24 hours at 20 degrees celcius. Synchronous L1s were fed for 3 hours with HB101 in liquid culture. They were then crosslinked in 2% formaldehyde in M9 for 30 minutes at 20 degrees celcius and quenched with 100mM Tris-HCl. They were washed in Homogenization buffer (50mM HEPES-KOH (pH 7.6), 0.5% NP-40, 140mM KCl, 1mM EDTA, 5mM DTT, 1mM PMSF, PI inhibitor cocktail (EMD Biosciences)) and frozen. Samples were then ground by mortar and pestle under liquid nitrogen and were resuspended in Homogenization buffer and snap frozen. Samples were then sheared using a sonicator.
Growth protocol
NG agar with E. coli HB101. Synchronous L1s fed in liquid culture for 3 hours with HB101
Extracted molecule
genomic DNA
Extraction protocol
Extracts from wild type N2 XX embryos were created as published in Pferdehirt et al. Briefly, gravid hermaphrodites were bleached and crosslinked with 2% formaldehyde in M9 at room temperature for 30 minutes. Crosslinking was quenched with 100mM Tris-HCl (pH 7.5). Embryos were resuspended and frozen in Homogenization buffer (50mM HEPES-KOH (pH 7.6), 0.5% NP-40, 140mM KCl, 1mM EDTA, 5mM DTT, 1mM PMSF). Samples were sheared with a sonicator. To collect fed L1 worms for ChIP-chip, gravid hermaphrodites were bleached and embryos were synchronized in M9 for 24 hours at 20 degrees celcius. Synchronous L1s were fed for 3 hours with HB101 in liquid culture. They were then crosslinked in 2% formaldehyde in M9 for 30 minutes at 20 degrees celcius and quenched with 100mM Tris-HCl. They were washed in Homogenization buffer (50mM HEPES-KOH (pH 7.6), 0.5% NP-40, 140mM KCl, 1mM EDTA, 5mM DTT, 1mM PMSF, PI inhibitor cocktail (EMD Biosciences)) and frozen. Samples were then ground by mortar and pestle under liquid nitrogen and were resuspended in Homogenization buffer and snap frozen. Samples were then sheared using a sonicator. Immunoprecipitations were performed with the appropriate antibody (as displayed in the beginning of each sample name) and were washed and processed as described in Pferdehirt et al.
Label
cy3
Label protocol
Sample labeling protocol provided and performed by the FHCRC Genomics Facility using standard NimbleGen protocol
Briefly, gravid hermaphrodites were bleached and embryos were synchronized in M9 for 24 hours at 20 degrees celcius. Synchronous L1s were fed for 3 hours with HB101 in liquid culture. They were then crosslinked in 2% formaldehyde in M9 for 30 minutes at 20 degrees celcius and quenched with 100mM Tris-HCl. They were washed in Homogenization buffer (50mM HEPES-KOH (pH 7.6), 0.5% NP-40, 140mM KCl, 1mM EDTA, 5mM DTT, 1mM PMSF, PI inhibitor cocktail (EMD Biosciences)) and frozen. Samples were then ground by mortar and pestle under liquid nitrogen and were resuspended in Homogenization buffer and snap frozen. Samples were then sheared using a sonicator.
Growth protocol
NG agar with E. coli HB101. Synchronous L1s fed in liquid culture for 3 hours with HB101
Extracted molecule
genomic DNA
Extraction protocol
Extracts from wild type N2 XX embryos were created as published in Pferdehirt et al. Briefly, gravid hermaphrodites were bleached and crosslinked with 2% formaldehyde in M9 at room temperature for 30 minutes. Crosslinking was quenched with 100mM Tris-HCl (pH 7.5). Embryos were resuspended and frozen in Homogenization buffer (50mM HEPES-KOH (pH 7.6), 0.5% NP-40, 140mM KCl, 1mM EDTA, 5mM DTT, 1mM PMSF). Samples were sheared with a sonicator. To collect fed L1 worms for ChIP-chip, gravid hermaphrodites were bleached and embryos were synchronized in M9 for 24 hours at 20 degrees celcius. Synchronous L1s were fed for 3 hours with HB101 in liquid culture. They were then crosslinked in 2% formaldehyde in M9 for 30 minutes at 20 degrees celcius and quenched with 100mM Tris-HCl. They were washed in Homogenization buffer (50mM HEPES-KOH (pH 7.6), 0.5% NP-40, 140mM KCl, 1mM EDTA, 5mM DTT, 1mM PMSF, PI inhibitor cocktail (EMD Biosciences)) and frozen. Samples were then ground by mortar and pestle under liquid nitrogen and were resuspended in Homogenization buffer and snap frozen. Samples were then sheared using a sonicator. Immunoprecipitations were performed with the appropriate antibody (as displayed in the beginning of each sample name) and were washed and processed as described in Pferdehirt et al.
Label
cy5
Label protocol
Sample labeling protocol provided and performed by the FHCRC Genomics Facility using standard NimbleGen protocol
Hybridization protocol
Hybridization protocol provided and performed by the FHCRC Genomics Facility using standard NimbleGen protocol
Scan protocol
Sample scan protocol provided and performed by the FHCRC Genomics Facility using standard NimbleGen protocol
Data processing
Log base 2 ratios of experimental/input were determined using SignalMap v2.5. For comparison with gene expression, the dynamic range of embryo and fed L1 ChIP- chip experimental values were normalized using STATA SE 9.2 (StataCorp LP).
An MLL/COMPASS subunit functions in the C. elegans dosage compensation complex to target X chromosomes for transcriptional regulation of gene expression
