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Sample GSM1064677

Query DataSets for GSM1064677

Status

Public on Jan 22, 2013

Title

Mouse testes INPUT

Sample type

SRA

Source name

testis

Organism

Mus musculus

Characteristics

tissue: testis

Extracted molecule

genomic DNA

Extraction protocol

Samples were obtained either as purified genomic DNA, as fresh-frozen samples or were dissected in-house and fresh-frozen. Samples were subjected to manual homogenisation followed by DNA purification by phenol chloroform extraction or using the QIAGEN 100/G genomic tip kit. For each individual Bio-CAP experiment, 25 μl of NeutrAvidin Agarose Resin (Thermo Scientific, 29200) was washed with BC100 buffer and then incubated with 50 μl of 0.5 μg/μl biotinylated hKDM2b-CxxC protein diluted in BC100 buffer for 1 hour at 4°C. The conjugated resin/CxxC protein was then washed with CAP100 buffer (12.5% glycerol, 0.1% Triton-x-100, 20 mM HEPES pH 7.9, 100 mM NaCl). Genomic DNA was sonicated to an average size of 150-250 bp using a Diagenode Bioruptor. Sonicated DNA was then diluted in CAP100 buffer to a concentration of 16 μg/ml and a 100 μl Input sample was retained. For each Bio-CAP experiment, 500 μl of diluted sonicated DNA, corresponding to approximately 8 μg of DNA, was added to the conjugated CxxC resin. The DNA and resin were incubated at 4°C for 1 hour with gentle mixing. The resin was then collected by centrifugation at 1500 x g for 3 minutes at 4°C, and the unbound flowthrough (FT) material was removed. The resin and any associated DNA was washed twice with 1 ml of CAP100 buffer, before the first elution was performed by adding 50 μl of CAP300 (12.5% glycerol, 0.1% Triton-x-100, 20 mM HEPES pH 7.9, 300 mM NaCl) to the resin and incubating at room temperature for 10 minutes. Following centrifugation a 50 μl elution fraction was carefully collected. The elution process was repeated using another 50 μl of CAP300 and the 300 mM elution fractions were pooled (giving a total volume of 100 μl). Subsequent elutions were performed in the same way using buffers with 500 mM, 700 mM and 1 M NaCl sequentially. Each 100 μl elution fraction, together with 100 μl of both the Input and FT samples, was purified using a PCR purification column (Qiagen) and DNA was eluted in a volume of 50 μl. The 700 mM and 1 M fractions were pooled for sequencing. Bio-CAP material was quantified using the High Sensitivity Qubit system (Invitrogen) and the fragmentation profile was assessed using a DNA HS Agilent 2200 TapeStation chip. Bio-CAP material was then prepared for Solexa sequencing using the NEBNextTM DNA Sample Prep Master Mix Set 1, according to manufacturer's specifications. All Bio-CAP experiments were performed in duplicate with matched input controls.
Next generation sequencing was performed using two Illumina sequencing platforms: Genome Analyser IIx and HiSeq Systems yielding 51bp single-end reads. Sequencing was carried out according to the manufacturer’s instructions.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 1000

Data processing

Bio-CAP reads were aligned to the appropriate reference genomes using Bowtie version 0.12.7 allowing a maximum of two mismatches in the entire read, and retaining only uniquely mapping reads
PCR duplicates were removed uisng Picard MarkDuplicates version 1.79
Peaks were called using MACS version 14 vs input
Peaks closer than 200bp apart were merged
Peaks from biological replicates were merged and only peaks found in both replicated were retained
Genome_build: anoCar2,danRer7,xenTro3,mm9,hg19, galGal3,ornAna1
Supplementary_files_format_and_content: bed files of replicated Bio-CAP peaks

Submission date

Jan 15, 2013

Last update date

May 15, 2019

Contact name

David Sims

E-mail(s)

david.sims@dpag.ox.ac.uk

Phone

+44 1865 82362

URL

http://www.cgat.org