Embryos were collected on grape agar plates from 5 g of Oregon R wild type adults over the course of 16 hours at 25oC.
Extracted molecule
genomic DNA
Extraction protocol
Approximately 500 mg of embryos were collected on grape agar plates from 5 g of Oregon R wild type adults over the course of 16 hours at 25oC. Embryos were dechorionated in 50% bleach for 90 seconds, rinsed thoroughly with distilled water, rinsed with 10ml of isopropanol then immediately plunged into a two-phase mixture of 30 ml n-heptane and 10 ml fixation buffer (50 mM HEPES pH 7.6, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA and 1.8% formaldehyde). Embryos were fixed for 15 minutes with vigorous agitation, centrifuged 1 minute at 500 rcf (25° C) to pellet, quenched for 1 minute with 125 mM Glycine (in PBS), then washed once with 50 ml PBT (PBS with 0.1% Triton X-100). The dry embryo cake was weighed, flash frozen and stored at -80° C. Fixed embryos were Dounce homogenized in 10 volumes of PBT using 20 strokes with a loose fitting pestle. The lysate was centrifuged for 1 minute at 400 rcf (4° C) to pellet unbroken embryos, and the supernatant was transferred to a new tube and centrifuged for 10 minutes at 1100 rcf (4° C) to obtain a crude nuclear pellet. The crude nuclear pellet was resuspended in 10 ml of cold Cell Lysis Buffer (50 mM HEPES pH7.6, 85 mM KCl, 0.5% NP-40, 1 mM PMSF, protease inhibitors) per mg of starting frozen fixed embryos, then Dounce homogenized using 20 strokes with a tight-fitting pestle. This homogenate was centrifuged for 4 minutes at 2000 rcf (4° C), resuspended in 1 ml cold Nuclear Lysis Buffer (50 mM Hepes pH 7.6, 10 mM EDTA, 0.5% N-lauroylsarcosine, 1 mM PMSF, protease inhibitors) per 750 mg starting frozen fixed embryos, and incubated at room temperature for 20 minutes. The nuclear lysate was diluted with an additional volume of cold Nuclear Lysis Buffer, then sonicated in a Diagenode Bioruptor with 8 30-second pulses at the medium intensity setting (2 minute rest between pulses) to obtain fragments of 200-600bp. Sonicated material was spun in a 4° C micro-centrifuge at maximum speed for 10 minutes, and the supernatant (ChIP-material) was stored at -80° C. For each sample, 75 µl ChIP-material was diluted into 1 ml IP buffer (0.5% Triton X-100, 2 mM EDTA, 20 mM Tris pH 8.0, 150 mM NaCl, 10% glycerol), pre-cleared with Protein-A agarose for 90 minutes, immuno-precipitated overnight with a concentration of antibody titrated to maximize signal:noise ratio, and incubated for 90 minutes with Protein-A agarose beads. Beads were washed extensively, bound material was eluted with Elution Buffer (1% SDS, 0.1 M NaHCO3), crosslinks were reversed, precipitated DNA was extracted once with phenol:chloroform:isoamyl alcohol and then ethanol precipitation. Precipitated DNA from 6-10 IPs was resuspended in water and pooled. RNA was degraded by 30 minute treatment with RNAse cocktail (Ambion), and DNA further purified by QIAquick PCR purification kit (QIAGEN).
Label
Cy5
Label protocol
25 ng immunoprecipitated or input DNA was amplified using the Sigma WGA2 Kit. Sample labeling was performed as described in the NimbleChip Array User’s Guide, except immediately following labeling samples were subjected to a second round of labeling consisting of incubation at 98° C for 10 minutes, 2 minute incubation on ice, addition of 2 µl Klenow exo- (NEB), and subsequent 2 hour incubation at 37° C.
Embryos were collected on grape agar plates from 5 g of Oregon R wild type adults over the course of 16 hours at 25oC.
Extracted molecule
genomic DNA
Extraction protocol
Approximately 500 mg of embryos were collected on grape agar plates from 5 g of Oregon R wild type adults over the course of 16 hours at 25oC. Embryos were dechorionated in 50% bleach for 90 seconds, rinsed thoroughly with distilled water, rinsed with 10ml of isopropanol then immediately plunged into a two-phase mixture of 30 ml n-heptane and 10 ml fixation buffer (50 mM HEPES pH 7.6, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA and 1.8% formaldehyde). Embryos were fixed for 15 minutes with vigorous agitation, centrifuged 1 minute at 500 rcf (25° C) to pellet, quenched for 1 minute with 125 mM Glycine (in PBS), then washed once with 50 ml PBT (PBS with 0.1% Triton X-100). The dry embryo cake was weighed, flash frozen and stored at -80° C. Fixed embryos were Dounce homogenized in 10 volumes of PBT using 20 strokes with a loose fitting pestle. The lysate was centrifuged for 1 minute at 400 rcf (4° C) to pellet unbroken embryos, and the supernatant was transferred to a new tube and centrifuged for 10 minutes at 1100 rcf (4° C) to obtain a crude nuclear pellet. The crude nuclear pellet was resuspended in 10 ml of cold Cell Lysis Buffer (50 mM HEPES pH7.6, 85 mM KCl, 0.5% NP-40, 1 mM PMSF, protease inhibitors) per mg of starting frozen fixed embryos, then Dounce homogenized using 20 strokes with a tight-fitting pestle. This homogenate was centrifuged for 4 minutes at 2000 rcf (4° C), resuspended in 1 ml cold Nuclear Lysis Buffer (50 mM Hepes pH 7.6, 10 mM EDTA, 0.5% N-lauroylsarcosine, 1 mM PMSF, protease inhibitors) per 750 mg starting frozen fixed embryos, and incubated at room temperature for 20 minutes. The nuclear lysate was diluted with an additional volume of cold Nuclear Lysis Buffer, then sonicated in a Diagenode Bioruptor with 8 30-second pulses at the medium intensity setting (2 minute rest between pulses) to obtain fragments of 200-600bp. Sonicated material was spun in a 4° C micro-centrifuge at maximum speed for 10 minutes, and the supernatant (ChIP-material) was stored at -80° C. For each sample, 75 µl ChIP-material was diluted into 1 ml IP buffer (0.5% Triton X-100, 2 mM EDTA, 20 mM Tris pH 8.0, 150 mM NaCl, 10% glycerol), pre-cleared with Protein-A agarose for 90 minutes, immuno-precipitated overnight with a concentration of antibody titrated to maximize signal:noise ratio, and incubated for 90 minutes with Protein-A agarose beads. Beads were washed extensively, bound material was eluted with Elution Buffer (1% SDS, 0.1 M NaHCO3), crosslinks were reversed, precipitated DNA was extracted once with phenol:chloroform:isoamyl alcohol and then ethanol precipitation. Precipitated DNA from 6-10 IPs was resuspended in water and pooled. RNA was degraded by 30 minute treatment with RNAse cocktail (Ambion), and DNA further purified by QIAquick PCR purification kit (QIAGEN).
Label
Cy3
Label protocol
25 ng immunoprecipitated or input DNA was amplified using the Sigma WGA2 Kit. Sample labeling was performed as described in the NimbleChip Array User’s Guide, except immediately following labeling samples were subjected to a second round of labeling consisting of incubation at 98° C for 10 minutes, 2 minute incubation on ice, addition of 2 µl Klenow exo- (NEB), and subsequent 2 hour incubation at 37° C.
Hybridization protocol
Hybridization and washing were performed as described in the NimbleChip Array User’s Guide.
Scan protocol
Arrays were scanned with an Agilent G2565CA scanner at 5 µm resolution, two-pass scanning, with manually adjusted gain settings.
Description
Chromatin immunoprecipitated with antibody raised against Rpb3
Data processing
Data were processed using Nimblescan software (Nimblegen) according to the manufacturer's instructions. For presentation, log2 ratios were converted to fold-enrichment values with fold enrichment of 1 representing the genome-wide average.
