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.
Growth protocol
sdc-2(y93) grown on NG plates suplimented with 100 µg/ml carbinicillin and 1 µg/ml IPTG seeded with E. coli HT115 (DE3) carrying an RNAi vector to sdc-2
Extracted molecule
genomic DNA
Extraction protocol
Extracts from wild type N2 XX, sdc-2mut (y93,RNAi) XX, sdc-2mut TY2222 XO, sdc-3mut TY2205 XO, dpy-27mut TY2470 XO, dpy-30mut TY1119 XX, and X:V fusion ypT28 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. 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 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.
Growth protocol
sdc-2(y93) grown on NG plates suplimented with 100 µg/ml carbinicillin and 1 µg/ml IPTG seeded with E. coli HT115 (DE3) carrying an RNAi vector to sdc-2
Extracted molecule
genomic DNA
Extraction protocol
Extracts from wild type N2 XX, sdc-2mut (y93,RNAi) XX, sdc-2mut TY2222 XO, sdc-3mut TY2205 XO, dpy-27mut TY2470 XO, dpy-30mut TY1119 XX, and X:V fusion ypT28 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. 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. No normalization.
An MLL/COMPASS subunit functions in the C. elegans dosage compensation complex to target X chromosomes for transcriptional regulation of gene expression
