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Sample GSM1517812

Query DataSets for GSM1517812

Status

Public on Oct 03, 2014

Title

WT_IC_RC12h.2011_11_30

Sample type

SRA

Source name

mycelia

Organism

Aspergillus fumigatus

Characteristics

strain: CBS 144.89

Treatment protocol

hypoxia, 1% oxygen, 5%CO2

Growth protocol

All strains are routinely grown in liquid glucose minimal media (LGMM) that contains 1% glucose, salt solution and trace minerals, at 37°C, under agitation (200RPM) in baffle flasks.

Extracted molecule

genomic DNA

Extraction protocol

After reverse crosslinking, samples were treated with 2.5 mg of RNase A and incubated for 30 minutes at room temperature. DNA was extracted either by using PCR purification kit (Qiagen) following the high pH protocol, or EtOH precipitation, with 50 mL as final volume. 5mL of sample was assayed on Qubit using high sensitivity dsDNA kit (Invitrogen). To check sonication, 10μL of IC was run on E-gel EX 2% agarose gel (Invitrogen) and fragment size ranged from 1kb to 100bp
fragmented ends were repaired, an adenine molecule was added to the repaired end, PAGE-purified adapters were added to the overhang, and the mixture amplified with primer 1.0 and primer 2.0 with an individual index tag to allow for multiplex of samples in a single lane. Samples were gel purified to obtain a size range between 200-400bp. Libraries were validated by real time PCR, concentration was determined with Qubit (Invitrogen) and integrity was checked with an Agilent Bioanalyzer.
76 bp paired end sequencing and four ChIP samples were indexed per lane on the Illumina HiSeq 2000.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2000

Description

replicate 3

Data processing

Illumina HiSeq fastq files were quality filtered using Trimmomatic-0.30 using parameters as in the paired end example in the trimmomatic manual, keeping only reads >= 36 bp and requiring each of the paired reads pass the filtering.
Trimmomatic quality filtered fastq reads were aligned to the Aspergillus_fumigatusa1163.CADRE.18.dna.toplevel.fa genome (http://fungi.ensembl.org/info/website/ftp/index.html) using bowtie2-2.1.0, keeping only concordantly mapped read pairs.
The concordantly aligned paired end bam files were subsampled using samtools-0.1.19 view -s option such that number of reads in input control approximately matched number of reads in ChIP sample within replicate.
Subsampled bam files were filtered to genearate deduplicated bam files using samtools-0.1.19 rmdup followed by picard-tools-1.93 MarkDuplicates with REMOVE_DUPLICATES=TRUE.
Deduplicated bam files for each replicate were combined to obtain a single input control bam file and single ChIP bam file.
The combined single ChIP bam file and combined single IC bam file were used as input for peak calling using macs2 version 2.0.10.20131216 (parameters: mfold 5,50; format bampe; keep-dup all; call-summits).
Genome_build: Aspergillus_fumigatusa1163.CADRE.18.dna.toplevel.fa (http://fungi.ensembl.org/info/website/ftp/index.html)
Supplementary_files_format_and_content: tab separated text file. The peaks file from macs2 with run details and called peak locations and scores.

Submission date

Oct 02, 2014

Last update date

May 15, 2019

Contact name

Bridget Marie Barker

E-mail(s)

[email protected]

Organization name

TGEN-North

Department

CFP