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Sample GSM2057956 Query DataSets for GSM2057956
Status Public on May 17, 2016
Title Ha-Ras mut tumour mouse 1 5mC profile
Sample type genomic
 
Channel 1
Source name Ha-Ras liver tumour
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)
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.
Label Cy5
Label protocol Amplification of DNA samples was carried out commercially by NimbleGen, Iceland (Cy5- (IP) or Cy3- (Input) labelled)
 
Channel 2
Source name Ha-Ras liver tumour_input
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)
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.
Label Cy3
Label protocol Amplification of DNA samples was carried out commercially by NimbleGen, Iceland (Cy5- (IP) or Cy3- (Input) labelled)
 
 
Hybridization protocol Labelled samples were applied commercially to a "Nimblegen 2.1M Deluxe promoter array" by Nimblegen, Iceland
Scan protocol Arrays were scanned commercially by Nimblegen, Iceland
Description HaRas_Tumour_1_mc
Data processing Normalised data after normalised within arrays (loess) and between arrays (sclae normalisation) resulting in log2(IP/input) scores for each probe
 
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
Organization name University of Edinburgh
Department MRC Human Genetics Unit
Lab Meehan
Street address Crewe Road
City Edinburgh
ZIP/Postal code EH4 2XU
Country United Kingdom
 
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

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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