cell type: Drosophila S2 Cells (Invitrogen) fraction: Pol II (Rpb3) IP DNA agent: 1 µM 20-hydroxyecdysone
Treatment protocol
Cells were treated for 24 hours with 1 µM 20-hydroxyecdysone before crosslinking and sonication.
Growth protocol
Drosophila S2 cells ( Invitrogen) were grown in Schneider’s media +10% FBS without additional supplementation.
Extracted molecule
genomic DNA
Extraction protocol
For ecdysone treatment experiments, Invitrogen S2 cells were diluted to a density of ~1E6 cells/ml and were grown in the presence of 1 µM 20-hyrodxyecdysone or vehicle for 24 hours. Cells were then resuspended by scraping, diluted 1:2 in M3 media lacking serum, crosslinked with 1% formaldehyde for 10 minutes, then quenched with glycine at a final concentration of 125 mM. Crosslinked cells were washed once with cold PBS, resuspended in 1 ml cold Sonication buffer (0.5% SDS, 20 mM Tris pH8.0, 2 mM EDTA, 0.5 mM EGTA, 0.5 mM PMSF, protease inhibitors) per 108 cells, incubated on ice for 10 minutes, then sonicated in a Diagenode Bioruptor with 12 30-second pulses, each pulse followed by a 2.5-minute rest, to obtain fragments of 200-600 bp. 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). For TFIIA experiments, DGRC S2 cells were grown to a density of ~5E6 cells/ml, then processed as described above except that cells were crosslinked for 30 minutes before quenching.
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.
Cells were treated for 24 hours with 1 µM 20-hydroxyecdysone before crosslinking and sonication.
Growth protocol
Drosophila S2 cells ( Invitrogen) were grown in Schneider’s media +10% FBS without additional supplementation.
Extracted molecule
genomic DNA
Extraction protocol
For ecdysone treatment experiments, Invitrogen S2 cells were diluted to a density of ~1E6 cells/ml and were grown in the presence of 1 µM 20-hyrodxyecdysone or vehicle for 24 hours. Cells were then resuspended by scraping, diluted 1:2 in M3 media lacking serum, crosslinked with 1% formaldehyde for 10 minutes, then quenched with glycine at a final concentration of 125 mM. Crosslinked cells were washed once with cold PBS, resuspended in 1 ml cold Sonication buffer (0.5% SDS, 20 mM Tris pH8.0, 2 mM EDTA, 0.5 mM EGTA, 0.5 mM PMSF, protease inhibitors) per 108 cells, incubated on ice for 10 minutes, then sonicated in a Diagenode Bioruptor with 12 30-second pulses, each pulse followed by a 2.5-minute rest, to obtain fragments of 200-600 bp. 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). For TFIIA experiments, DGRC S2 cells were grown to a density of ~5E6 cells/ml, then processed as described above except that cells were crosslinked for 30 minutes before quenching.
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.
