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| Status |
Public on Oct 11, 2014 |
| Title |
T1 leg disc CTCF-ChIP Rep_1 |
| Sample type |
genomic |
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| Channel 1 |
| Source name |
Leg 1 Preimmune Rep 1
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| Organism |
Drosophila melanogaster |
| Characteristics |
Stage: late 3rd instar larva
strain: Oregon-R
chip antibody: pre-immune serum (Holohan et al., 2007 Plos Genet 3: e112. doi:10.1371/journal.pgen.0030112.)
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| Treatment protocol |
Dissected head segments of late 3rd instar larvae were inverted and fixed with 2% formaldehyde in PBS for 20 min at room temperature. These were washed with twice with PBS/125 mM Glycine/0.01% Triton X-100 followed by a single wash with PBS and then with PBS containing 1% protease inhibitor cocktail (Sigma, P8340). The T1 and T3 leg imaginal discs were then dissected, snap frozen in liquid nitrogen and stored at -80°C prior to use. Approximately 150 leg discs were combined in PBS/0.01% Triton X-100 and centrifuged in a microfuge at 1200 rpm for 1 min. The discs were resuspended in 20 μl cell lysis buffer (5 mM PIPES pH 8, 85 mM KCl, 0.5% NP-40) containing 1% protease inhibitor cocktail and homogenised using a motorised pestle at 2 min intervals for 8 min. After a brief microfuge centrifugation (13,200 rpm, 10 sec), the pellet was resuspended in 300 µl Nuclear Lysis Buffer (50 mM Tris.HCl pH 8.1, 10 mM EDTA.Na2, 1% SDS) with protease inhibitors and incubated for 20 min at room temperature. The extracts were sonicated in a Bioruptor (Diagenode) at high setting for 4 min 15 sec (30 sec “ON”, 30 sec “OFF” cycle), producing 0.5 to 3.0 kb sized fragments. 100 μl aliquots of chromatin extracts were flash frozen in liquid nitrogen and then stored at -80°C prior to use.
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| Growth protocol |
Flies were grown on standard medium at 18°C.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin immunopurification was performed as described by Birch-Machin (2005). 100 µl aliquots of chromatin were pre-cleared with 13 µl blocked S. aureus cells (SAC) and mixed with 200 µl of IP dilution buffer (16.7mM Tris.HCl pH 8, 167mM NaCl, 1% EDTA, 1.1% Triton X-100, 0.01% SDS) with protease inhibitors. 2 µl of antibody was added and incubated on a roller overnight at 4°C. Then 13 µl of SAC was added to each IP reaction and the samples were incubated for 35 min at 4°C on a roller. The mixture was centrifuged in a microfuge at 13,200 rpm at room temperature and the pellets were washed successively with 1 ml each of Low Salt Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA. Na2 pH 8.0, 20 mM Tris.HCl pH 8, 150 mM NaCl), High Salt Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA.Na2 pH8, 20 mM Tris.HCl pH 8, 500mM NaCl), LiCl Buffer (0.25 M LiCl, 1% NP40, 1% NaDeoxycholate, 1 mM EDTA. Na2 pH 8.0, 10 mM Tris.HCl pH 8.0) and twice with TE buffer pH 8.0, for 5 min at 4°C on roller for each solution. The immune-precipitated chromatin was then eluted twice from the SAC pellet with 300 μl of IP elution buffer (50 mM NaHCO3, 1% SDS) by vigorously vortexing for 15 min at room temperature. One μl of RNase A (Sigma, R4642) and 24.3 μl of 4M NaCl (0.3 M final concentration) were then added to the eluate and the mixture was incubated for 4h at 65°C, to reverse the cross-linking. The DNA was then precipitated by adding 812 μl of 100% ethanol and incubating overnight at -20°C. The samples were centrifuged in a microfuge at 4°C for 20 min and the pellets were air dried for 1 h at room temperature. The pellets were resuspended in 100 μl TE buffer followed by the addition of 25 μl of 5X PK buffer (50 mM Tris HCl pH 7.5, 25 mM EDTA.Na2 pH 8, 1.25% SDS) and 1.5 μl of 20 mg/mL Proteinase K, incubated at 45°C for 2 h and purified using the QIAquick PCR Purification Kit (Qiagen, 28104). The DNA was eluted in 30 μl of buffer EB and stored at -20°C until use.
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| Label |
Cy3
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| Label protocol |
5 µl each of CTCF-ChIP and control ChIP DNA from T1 and T3 leg discs obtained from Oregon R larvae were amplified using GenomePlex Single Cell Whole Genome Amplification Kit (Sigma-Aldrich WGA4) according to manufacturer’s instructions. The samples were amplified for 21 cycles and the amplified DNA purified using the QIAquick PCR Purification Kit. 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).
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| Channel 2 |
| Source name |
Leg 1 CTCF Rep 1
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| Organism |
Drosophila melanogaster |
| Characteristics |
Stage: late 3rd instar larva
strain: Oregon-R
chip antibody: anti-CTCF-C1 (Holohan et al., 2007 Plos Genet 3: e112. doi:10.1371/journal.pgen.0030112.)
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| Treatment protocol |
Dissected head segments of late 3rd instar larvae were inverted and fixed with 2% formaldehyde in PBS for 20 min at room temperature. These were washed with twice with PBS/125 mM Glycine/0.01% Triton X-100 followed by a single wash with PBS and then with PBS containing 1% protease inhibitor cocktail (Sigma, P8340). The T1 and T3 leg imaginal discs were then dissected, snap frozen in liquid nitrogen and stored at -80°C prior to use. Approximately 150 leg discs were combined in PBS/0.01% Triton X-100 and centrifuged in a microfuge at 1200 rpm for 1 min. The discs were resuspended in 20 μl cell lysis buffer (5 mM PIPES pH 8, 85 mM KCl, 0.5% NP-40) containing 1% protease inhibitor cocktail and homogenised using a motorised pestle at 2 min intervals for 8 min. After a brief microfuge centrifugation (13,200 rpm, 10 sec), the pellet was resuspended in 300 µl Nuclear Lysis Buffer (50 mM Tris.HCl pH 8.1, 10 mM EDTA.Na2, 1% SDS) with protease inhibitors and incubated for 20 min at room temperature. The extracts were sonicated in a Bioruptor (Diagenode) at high setting for 4 min 15 sec (30 sec “ON”, 30 sec “OFF” cycle), producing 0.5 to 3.0 kb sized fragments. 100 μl aliquots of chromatin extracts were flash frozen in liquid nitrogen and then stored at -80°C prior to use.
|
| Growth protocol |
Flies were grown on standard medium at 18°C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin immunopurification was performed as described by Birch-Machin (2005). 100 µl aliquots of chromatin were pre-cleared with 13 µl blocked S. aureus cells (SAC) and mixed with 200 µl of IP dilution buffer (16.7mM Tris.HCl pH 8, 167mM NaCl, 1% EDTA, 1.1% Triton X-100, 0.01% SDS) with protease inhibitors. 2 µl of antibody was added and incubated on a roller overnight at 4°C. Then 13 µl of SAC was added to each IP reaction and the samples were incubated for 35 min at 4°C on a roller. The mixture was centrifuged in a microfuge at 13,200 rpm at room temperature and the pellets were washed successively with 1 ml each of Low Salt Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA. Na2 pH 8.0, 20 mM Tris.HCl pH 8, 150 mM NaCl), High Salt Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA.Na2 pH8, 20 mM Tris.HCl pH 8, 500mM NaCl), LiCl Buffer (0.25 M LiCl, 1% NP40, 1% NaDeoxycholate, 1 mM EDTA. Na2 pH 8.0, 10 mM Tris.HCl pH 8.0) and twice with TE buffer pH 8.0, for 5 min at 4°C on roller for each solution. The immune-precipitated chromatin was then eluted twice from the SAC pellet with 300 μl of IP elution buffer (50 mM NaHCO3, 1% SDS) by vigorously vortexing for 15 min at room temperature. One μl of RNase A (Sigma, R4642) and 24.3 μl of 4M NaCl (0.3 M final concentration) were then added to the eluate and the mixture was incubated for 4h at 65°C, to reverse the cross-linking. The DNA was then precipitated by adding 812 μl of 100% ethanol and incubating overnight at -20°C. The samples were centrifuged in a microfuge at 4°C for 20 min and the pellets were air dried for 1 h at room temperature. The pellets were resuspended in 100 μl TE buffer followed by the addition of 25 μl of 5X PK buffer (50 mM Tris HCl pH 7.5, 25 mM EDTA.Na2 pH 8, 1.25% SDS) and 1.5 μl of 20 mg/mL Proteinase K, incubated at 45°C for 2 h and purified using the QIAquick PCR Purification Kit (Qiagen, 28104). The DNA was eluted in 30 μl of buffer EB and stored at -20°C until use.
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| Label |
Cy5
|
| Label protocol |
5 µl each of CTCF-ChIP and control ChIP DNA from T1 and T3 leg discs obtained from Oregon R larvae were amplified using GenomePlex Single Cell Whole Genome Amplification Kit (Sigma-Aldrich WGA4) according to manufacturer’s instructions. The samples were amplified for 21 cycles and the amplified DNA purified using the QIAquick PCR Purification Kit. 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).
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| Hybridization protocol |
Resuspend samples in 12.3 µl water. For each hybridisation, combine 29.5µl 2X Hybridization Buffer, 11.8µl Hybridization Component A and 1.2µl Alignment Oligo (NimbleGen Hybridization Kit) and add 31.7µl to each hybridisation sample. Incubate samples at 95°C for 5 minutes, 42°C for 5 minutes and load 41 µl onto the array. The hybridisation is 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 16 hours. Perform post hybridisation washes according to the NimbleGen Wash Buffer Kit instructions.
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| Scan protocol |
Arrays are scanned at 5 µm resolution with a GenePix 4000B (Axon) dual laser scanner at 100% Power, with Lines to average = 1 and Focus position = 0 at their respective optimal PMT gains. Signal for Cy3 and Cy5 channels are balanced using the histogram view in the GenePix software. Images are saved as Single-image TIFF.
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| Description |
T1_CTCF.txt
T_1CTPRE.window_scores.sgr
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| Data processing |
The scanned images were processed using NimbleScan software to generate pair files which were then converted to chromatin enrichment profiles using the TiMAT pipeline (http://bdtnp.lbl.gov/TiMAT/). Samples were median scaled at 500, quantile normalised (ChIP and Pre-immune ChIP samples together), window smoothed using a window size of 500 bp and a minimum number of oligos per window of 4.
quantile normalised log2 ratio (sample/control)
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| Submission date |
Oct 10, 2014 |
| Last update date |
Oct 11, 2014 |
| Contact name |
Robert A.H. White |
| E-mail(s) |
rw108@cam.ac.uk
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| Organization name |
University of Cambridge
|
| Department |
Physiology, Development and Neuroscience
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| Lab |
White Lab
|
| Street address |
Downing Street
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| City |
Cambridge |
| ZIP/Postal code |
CB2 3DY |
| Country |
United Kingdom |
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| Platform ID |
GPL15641 |
| Series (1) |
| GSE62234 |
CTCF binding profiles in Drosophila melanogaster 3rd instar larva T1 and T3 leg discs |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM1523421_467386_CTPRE_532.pair.gz |
32.7 Mb |
(ftp)(http) |
PAIR |
| GSM1523421_467386_CTPRE_635.pair.gz |
32.6 Mb |
(ftp)(http) |
PAIR |
| Processed data are available on Series record |
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