GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM1833397

Query DataSets for GSM1833397

Status

Public on Oct 01, 2015

Title

Acar_Hindlimb_Input_DNA

Sample type

SRA

Source name

embryonic hindlimb

Organism

Anolis carolinensis

Characteristics

tissue: embryonic hindlimb
developmental stage: TS7/8
genotype: wild type
chip antibody: none

Growth protocol

Normal gestation

Extracted molecule

genomic DNA

Extraction protocol

After cross-linking in 1% formaldehyde in PBS for 30 minutes, tissues were rinsed and treated with trypsin for 5 minutes to generate a single cell suspension. Samples were then pre-disrupted with a Branson 450 Sonifier, and then sheared with a Bioruptor (Diagenode) to generate a chromatin size range of 200–600 bp. PureProteome Protein G Magnetic Beads (Millipore) were pre-incubated with H3k27ac antibody before incubating overnight with 100-200 μg of sheared chromatin. After washing, immune complexes were eluted from the beads, and protein-DNA crosslinks were reversed by incubating at 65˚C overnight. After treatment with RNase followed by proteinase K, samples were purified with the QIAquick PCR Purification Kit (Qiagen).
ChIP and input chromatin control libraries were produced using the NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina (New England BioLabs) as directed by the manufacturer with Illumina-compatible adapters. Libraries were sequenced with 50 bp single-end runs on an Illumina HiSeq 2000 at the HudsonAlpha Institute for Biotechnology.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2000

Data processing

Basecalls were performed CASAVA version 1.8.2.
Illumina reads were aligned to the mouse genome (mm9) or green anole genome (anoCar2) with Bowtie version 1.1.0 with the options "--best --sam -t -l 28 -n 3 -m 1".
ChIP-Seq data quality and fragment size was assessed from aligned reads for each sample using phantompeaktools (https://code.google.com/p/phantompeakqualtools/).
Regions of H3K27ac enrichment were determined using macs2 (version 2.0.10.20130501) with the options “--bdg --SPMR --nomodel --qvalue=0.05”. Extension size was determined for each sample based on the cross-correlation profile of the aligned reads.
BigWig files of H3k27ac signal visualized as fold enrichment compared to background were generated from treatment and control bedGraph files using macs2 bdgcmp with the options " --tdepth=1 --cdepth=1 --method=FE". BedGraph files were converted to BigWig using BEDtools slopBed command and the bedClip and bedGraphToBigWig from UCSC Kent Utilities as implemented in the bdg2bw script by Tao Liu (https://gist.github.com/taoliu/2469050).
Genome_build: mm9 (Mus musculus); anoCar2 (Anolis carolinensis)
Supplementary_files_format_and_content: BigWig files were generated from treatment and control signal files created by macs2 and represent the fold enrichment of the ChIP sample versus the input DNA control. Bed files were created from the first 6 columns of the narrowPeak format files created by macs2 and represent regions with significant H3K27ac enrichment compared to background signal from input DNA controls with a qvalue cutoff of 0.05.

Submission date

Jul 27, 2015

Last update date

May 15, 2019

Contact name

Carlos Rafael Infante

E-mail(s)

cinfante@uga.edu

Organization name

University of Georgia

Department

Genetics