cell line: DmD8 treatment: Notch stimulation time point: 60 min chip antibody: Pol II (Ser 2 and Ser 5 phosphorylated) antibody antibody vendor: Abcam antibody catalog #: ab5408
Treatment protocol
Notch signalling was initiated by replacing cell media with 2mM EDTA in PBS for 5 minutes. The EDTA was then washed out using normal culture media and the cells were collected after 60 minutes.
Growth protocol
DmD8 cells were cultured in Schneiders medium (Invitrogen) supplemented with 10% FBS (Sigma), 5ug/ml insulin (Sigma) and 5% penicillin / streptomycin (Sigma) according to standard protocols.
Extracted molecule
genomic DNA
Extraction protocol
Chromatin was crosslinked by fixing cells for 10 minutes with 1% formaldehyde (Sigma) at room temperature before addition of Glycine (Sigma) (0.2M final concentration) to stop the crosslinking reaction. Cells were then detached from plates and washed twice with PBS, keeping samples at 4°C at all times. Chromatin was released from cells by incubation with nuclear lysis buffer (50mM Tris-HCl pH 8.1, 10mM EDTA, 1% SDS, 1X complete protease inhibitor (Roche)) for 10 minutes on ice. Crosslinked chromatin was fragmented by sonication to an average length of roughly 500bp. Chromatin was then precleared by addition of rabbit IgG and protein G agarose beads (Santa Cruz Biotechnology) in IP dilution buffer (IPDB) (20mM Tris-HCl pH 8.1, 150mM NaCl, 2mM EDTA, 1% Triton X-100, 0.01% SDS, 1X complete protease inhibitor (Roche)) and incubation at 4°C for 5 hours with gentle agitation. 1µg Pol II (Ser 2 or Ser 5 phosphorylated) antibody (Abcam cat. no. ab5408) per sample was preincubated with protein G agarose beads in IPDB for 5 hours at 4°C with gentle agitation. Precleared chromatin was precipitated by addition of protein G agarose beads with prebound Pol II (Ser 2 or Ser 5 phosphorylated) antibody and incubation at 4°C overnight with gentle agitation. Beads with chromatin bound were washed twice with wash buffer 1 (20mM Tris-HCl pH 8.1, 50mM NaCl, 2mM EDTA, 1% Triton X-100, 0.1% SDS), twice with wash buffer 2 (10mM Tris-HCl pH 8.1, 250mM LiCl, 1mM EDTA, 1% NP-40, 1% deoxycholic acid) and once with TE pH 8.0. Chromatin was then eluted from the agarose beads by incubation with elution buffer (100mM NaHCO3, 1% SDS) for 10 minutes with vigorous shaking before decrosslinking (of precipitated samples and total input) by incubating at 65°C for 5 hours with NaCl (0.27M final concentration). Remaining proteins were removed by incubating with proteinase-K at 55°C overnight. DNA was then purified by phenol/chloroform extraction and ethanol precipitation. Binding relative to input was quantified by qPCR. For array analysis, DNA fragments and total input samples were amplified by ligation mediated PCR and labelled before hybridisation to tiling arrays.
Label
Cy5
Label protocol
In duplicate reactions: 1 μg of amplified DNA with 30 µl 2.5X Random Primers Solution (BioPrime DNA labelling kit, Life Technologies) was incubated at 100°C for 5 minutes and then cooled on ice. The samples were labelled as technical dye-swap replicates using the BioPrime DNA labelling kit (Life Technologies) in the presence of 4.5nmol fluorescently labelled Cy3- or Cy5-dCTP (GE Healthcare) and 72 U of Klenow at 37°C. After 2 hours the reactions were stopped with 7.5 µl Stop Buffer (BioPrime DNA labelling kit, Life Technologies). Duplicate reactions were pooled into single tubes and then precipitated with 165 µl Isopropanol and 0.2M sodium chloride and washed with cold 80% ethanol. The DNA was resuspened in 50 µl water and quantified on a Nanodrop spectrophotometer (Thermo Scientific).
cell line: DmD8 treatment: Notch stimulation time point: 60 min chip antibody: none
Treatment protocol
Notch signalling was initiated by replacing cell media with 2mM EDTA in PBS for 5 minutes. The EDTA was then washed out using normal culture media and the cells were collected after 60 minutes.
Growth protocol
DmD8 cells were cultured in Schneiders medium (Invitrogen) supplemented with 10% FBS (Sigma), 5ug/ml insulin (Sigma) and 5% penicillin / streptomycin (Sigma) according to standard protocols.
Extracted molecule
genomic DNA
Extraction protocol
Chromatin was crosslinked by fixing cells for 10 minutes with 1% formaldehyde (Sigma) at room temperature before addition of Glycine (Sigma) (0.2M final concentration) to stop the crosslinking reaction. Cells were then detached from plates and washed twice with PBS, keeping samples at 4°C at all times. Chromatin was released from cells by incubation with nuclear lysis buffer (50mM Tris-HCl pH 8.1, 10mM EDTA, 1% SDS, 1X complete protease inhibitor (Roche)) for 10 minutes on ice. Crosslinked chromatin was fragmented by sonication to an average length of roughly 500bp. Chromatin was then precleared by addition of rabbit IgG and protein G agarose beads (Santa Cruz Biotechnology) in IP dilution buffer (IPDB) (20mM Tris-HCl pH 8.1, 150mM NaCl, 2mM EDTA, 1% Triton X-100, 0.01% SDS, 1X complete protease inhibitor (Roche)) and incubation at 4°C for 5 hours with gentle agitation. 1µg Pol II (Ser 2 or Ser 5 phosphorylated) antibody (Abcam cat. no. ab5408) per sample was preincubated with protein G agarose beads in IPDB for 5 hours at 4°C with gentle agitation. Precleared chromatin was precipitated by addition of protein G agarose beads with prebound Pol II (Ser 2 or Ser 5 phosphorylated) antibody and incubation at 4°C overnight with gentle agitation. Beads with chromatin bound were washed twice with wash buffer 1 (20mM Tris-HCl pH 8.1, 50mM NaCl, 2mM EDTA, 1% Triton X-100, 0.1% SDS), twice with wash buffer 2 (10mM Tris-HCl pH 8.1, 250mM LiCl, 1mM EDTA, 1% NP-40, 1% deoxycholic acid) and once with TE pH 8.0. Chromatin was then eluted from the agarose beads by incubation with elution buffer (100mM NaHCO3, 1% SDS) for 10 minutes with vigorous shaking before decrosslinking (of precipitated samples and total input) by incubating at 65°C for 5 hours with NaCl (0.27M final concentration). Remaining proteins were removed by incubating with proteinase-K at 55°C overnight. DNA was then purified by phenol/chloroform extraction and ethanol precipitation. Binding relative to input was quantified by qPCR. For array analysis, DNA fragments and total input samples were amplified by ligation mediated PCR and labelled before hybridisation to tiling arrays.
Label
Cy3
Label protocol
In duplicate reactions: 1 μg of amplified DNA with 30 µl 2.5X Random Primers Solution (BioPrime DNA labelling kit, Life Technologies) was incubated at 100°C for 5 minutes and then cooled on ice. The samples were labelled as technical dye-swap replicates using the BioPrime DNA labelling kit (Life Technologies) in the presence of 4.5nmol fluorescently labelled Cy3- or Cy5-dCTP (GE Healthcare) and 72 U of Klenow at 37°C. After 2 hours the reactions were stopped with 7.5 µl Stop Buffer (BioPrime DNA labelling kit, Life Technologies). Duplicate reactions were pooled into single tubes and then precipitated with 165 µl Isopropanol and 0.2M sodium chloride and washed with cold 80% ethanol. The DNA was resuspened in 50 µl water and quantified on a Nanodrop spectrophotometer (Thermo Scientific).
Hybridization protocol
34 µg labeled sample DNA and 34 µg labeled control DNA were combined, dried in a speed vac with medium heat and resuspended in 12.3 µl water. For each hybridisation, 29.5µl 2X Hybridization Buffer, 11.8µl Hybridization Component A and 1.2µl Alignment Oligo (NimbleGen Hybridization Kit) were combined and 31.7µl added to each hybridisation sample. Samples were incubated at +95°C for 5 minutes, 42°C for 5 minutes and 41 µl loaded onto the array. The hybridisation was performed to NimbleGen D. melanogaster ChIP-chip 2.1M Whole-Genome Tiling Arrays in the Nimbelegen hybridisation station at 42°C (mix mode B) for 18 hours. Post-hybridisation washes were performed according to the NimbleGen Wash Buffer Kit instructions.
Arrays were processed using Nimblegen's standard protocol for Nimblescan 2.4 ChIP data extraction. Quantile normalization was applied to the 3 arrays at each of the 7 time points.
ChIP-chip time-course from DmD8 cells with Pol II (Ser 2 and Ser 5 phosphorylated) antibody after 0, 10, 20, 30 40, 60 and 100 minutes Notch activation
