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Status |
Public on May 17, 2016 |
Title |
Ha-Ras mut tumour mouse 1 5mC profile |
Sample type |
genomic |
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Channel 1 |
Source name |
Ha-Ras liver tumour
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Organism |
Mus musculus |
Characteristics |
strain background: C3H/HeJ gender: Male genotype/variation: Wild type tissue type: DEN only exposed mouse liver resulting in liver tumour antibody: 5-mc - Eurogentec(cat# BI-MECY-1000)
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Extracted molecule |
genomic DNA |
Extraction protocol |
Genomic DNA (5mC-IP - 2.5 µg in 450 µl TE), sonicated to yield a fragment distribution of approximately 300-1000 bp, was denatured by incubation at 100°C for 10 min. Samples were rapidly chilled on wet-ice. At this point, 45 µl (10%) of denatured sample was removed and saved as input, and 45 µl of 10X IP buffer (100 mM Na-Phosphate pH 7.0 (mono and dibasic), 1.4 M NaCl, 0.5 % Triton X-100) and 1 µg of 5mC (ActiveMotif; #39769) antibody were added to the remaining sample. Samples were incubated at 4C with gentle agitation overnight. Then, 40 µl of magnetic beads (Dynabeads® Protein G, Invitrogen, UK) in 1X IP buffer were added to each sample to allow magnetic separation of the antibody from the unbound DNA using a magnetic tube rack. Samples were incubated at 4C for 1 hr with gentle agitation. Beads were collected with a magnetic rack and washed with 1000 µl of 1X IP buffer at RT for 10 min with gentle agitation; washing was repeated three times. Beads were collected with a magnetic rack and re-suspended in 250 µl of digestion buffer (50 mM Tris pH 8.0, 10 mM EDTA , 0.5 % SDS) followed by addition of 10 µl of proteinase K (20 mg/ml; Roche Applied Science, Mannheim, Germany) and incubation at 52C for 1.5 hr with constant shaking (≥800 rpm). Finally, beads were removed using a magnetic rack and DNA was purified from the remaining sample using a QIAquick PCR Purification Kit (QIAGEN,CA, USA), eluting in a final volume of 40 µl dH2O. Inputs were also purified using a QIAquick PCR Purification Kit and eluted in 40 µl dH2O. Subsequently, 10 ng of input and IP DNA was subjected to whole genome amplification (WGA) using the GenomePlex® Complete Whole Genome Amplification Kit (Sigma-Aldrich, UK) as per the manufacturer's instructions. Amplified DNA was run on a 1.2% agarose gel to confirm consistency of fragment size between samples.
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Label |
Cy5
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Label protocol |
Amplification of DNA samples was carried out commercially by NimbleGen, Iceland (Cy5- (IP) or Cy3- (Input) labelled)
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Channel 2 |
Source name |
Ha-Ras liver tumour_input
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Organism |
Mus musculus |
Characteristics |
strain background: C3H/HeJ gender: Male genotype/variation: Wild type tissue type: DEN only exposed mouse liver resulting in liver tumour antibody: none (Input)
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Extracted molecule |
genomic DNA |
Extraction protocol |
Genomic DNA (5mC-IP - 2.5 µg in 450 µl TE), sonicated to yield a fragment distribution of approximately 300-1000 bp, was denatured by incubation at 100°C for 10 min. Samples were rapidly chilled on wet-ice. At this point, 45 µl (10%) of denatured sample was removed and saved as input, and 45 µl of 10X IP buffer (100 mM Na-Phosphate pH 7.0 (mono and dibasic), 1.4 M NaCl, 0.5 % Triton X-100) and 1 µg of 5mC (ActiveMotif; #39769) antibody were added to the remaining sample. Samples were incubated at 4C with gentle agitation overnight. Then, 40 µl of magnetic beads (Dynabeads® Protein G, Invitrogen, UK) in 1X IP buffer were added to each sample to allow magnetic separation of the antibody from the unbound DNA using a magnetic tube rack. Samples were incubated at 4C for 1 hr with gentle agitation. Beads were collected with a magnetic rack and washed with 1000 µl of 1X IP buffer at RT for 10 min with gentle agitation; washing was repeated three times. Beads were collected with a magnetic rack and re-suspended in 250 µl of digestion buffer (50 mM Tris pH 8.0, 10 mM EDTA , 0.5 % SDS) followed by addition of 10 µl of proteinase K (20 mg/ml; Roche Applied Science, Mannheim, Germany) and incubation at 52C for 1.5 hr with constant shaking (≥800 rpm). Finally, beads were removed using a magnetic rack and DNA was purified from the remaining sample using a QIAquick PCR Purification Kit (QIAGEN,CA, USA), eluting in a final volume of 40 µl dH2O. Inputs were also purified using a QIAquick PCR Purification Kit and eluted in 40 µl dH2O. Subsequently, 10 ng of input and IP DNA was subjected to whole genome amplification (WGA) using the GenomePlex® Complete Whole Genome Amplification Kit (Sigma-Aldrich, UK) as per the manufacturer's instructions. Amplified DNA was run on a 1.2% agarose gel to confirm consistency of fragment size between samples.
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Label |
Cy3
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Label protocol |
Amplification of DNA samples was carried out commercially by NimbleGen, Iceland (Cy5- (IP) or Cy3- (Input) labelled)
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Hybridization protocol |
Labelled samples were applied commercially to a "Nimblegen 2.1M Deluxe promoter array" by Nimblegen, Iceland
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Scan protocol |
Arrays were scanned commercially by Nimblegen, Iceland
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Description |
HaRas_Tumour_1_mc
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Data processing |
Normalised data after normalised within arrays (loess) and between arrays (sclae normalisation) resulting in log2(IP/input) scores for each probe
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Submission date |
Feb 09, 2016 |
Last update date |
May 17, 2016 |
Contact name |
John Paterson Thomson |
E-mail(s) |
john.thomson@igmm.ed.ac.uk
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Organization name |
University of Edinburgh
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Department |
MRC Human Genetics Unit
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Lab |
Meehan
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Street address |
Crewe Road
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City |
Edinburgh |
ZIP/Postal code |
EH4 2XU |
Country |
United Kingdom |
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Platform ID |
GPL17878 |
Series (2) |
GSE77727 |
IP of 5-methylcytosine (5-mc) enriched DNA fragments from control, PB treated mouse livers, resulting Ctnnb1 mutated PB liver tumours and PB minus Ha-Ras mutate liver tumour |
GSE77731 |
Profiling of epigenetic and transcriptomic landscapes in normal mouse liver, phenobarbital exposed mouse livers and mouse liver tumours |
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Supplementary file |
Size |
Download |
File type/resource |
GSM2057956_Hras_Tum_mc_IP.pair.gz |
37.7 Mb |
(ftp)(http) |
PAIR |
GSM2057956_Hras_Tum_mc_input.pair.gz |
37.9 Mb |
(ftp)(http) |
PAIR |
Processed data are available on Series record |
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