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Sample GSM5747749 Query DataSets for GSM5747749
Status Public on Dec 31, 2023
Title ChIP Tbx3 FL bud E9.75-E10.25 replicate 2
Sample type SRA
 
Source name forelimb buds
Organism Mus musculus
Characteristics strain: Swiss albino
tissue: forelimb buds
developmental stage: E9.75-E10.25 (28-32 somites)
genotype: Tbx33XF/3XF
chip antibody: Mouse monoclonal M2 anti-FLAG antibodies (Sigma, F1804)
Treatment protocol Chromatin immunoprecipitation was performed as previously described (PMID: 27974206): Two replicates were generated and analysed: Per replicate 70 Tbx33XF/3XF forelimb buds were cross-linked with 1% formaldehyde at room temperature for 10 min. Chromatin was sonicated to obtain DNA fragments with an average size ranging between 100-600 bp. Protein A and G Dynabeads (Invitrogen 10001D, 10003D) were incubated overnight at 4oC with 5µg with 5 µg of anti-flagM2 antibody. Subsequently, the sonicated chromatin complexes were incubated with the M2 anti-FLAG antibodies coupled to beads overnight at 4oC. The immuno-complexes were then sequentially washed to remove non-specific complexes.
Extracted molecule genomic DNA
Extraction protocol The protein/DNA complexes were eluted using an SDS buffer (1% SDS, 50 mM Tris pH 8.0, 10 mM EDTA) at 65oC overnight. Samples were treated for 15 min RT with RNase A and then with Proteinase K for one hour at 56oC. Finally, the DNA was purified using MicroChip Diapure columns (Diagenode).
Libraries were prepared using MicroPlex Library preparation kit V2 (Diagenode) according to the manufactures instructions and 80 bp paired-end reads were sequenced using the Illumina Nextseq 500.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NextSeq 500
 
Description For Tbx3 ChIP-seq, a 3XFlag epitope tag was inserted into the carboxy-terminal end of the endogenous Tbx3 coding region.
Data processing High quality reads for Tbx3 samples were aligned on mm10 reference using Bowtie v2.2.9.
Mapped reads were filtered for duplicates using Picard (BroadInstitute 2015). Filtered bam files were subsequently sorted and indexed using Samtools (Li et al., 2009).
Peaks were called using Masc2 (Zhang et al. 2008) with settings -g mm -p 1e-3 --nomodel --extsize --call-summits -B --SPMR.
Evidences from these replicates were combined using MSPC (PMID: 25957351) with -r biological -s 1E-5 -W 1E-2 settings.
Normalized profiles were generated in wig/bigWig format to visualize on UCSC genome browser.
Genome_build: mm10
Supplementary_files_format_and_content: bigWig files to visualize signal; bigBed files with called peak regions.
 
Submission date Dec 22, 2021
Last update date Dec 31, 2023
Contact name Aimee Zuniga
E-mail(s) devgenlab-dbm@unibas.ch
Organization name University of Basel
Department Department of Biomedicine
Lab Developmental Genetics
Street address Mattenstrasse, 28 4058
City Basel
ZIP/Postal code 4058
Country Switzerland
 
Platform ID GPL19057
Series (2)
GSE192484 Identification of TBX3 and shared HAND2-TBX3 target genes in early mouse limb buds [ChIP-seq]
GSE192486 Identification of TBX3 and shared HAND2-TBX3 target genes in early mouse limb buds
Relations
BioSample SAMN24300609
SRA SRX13488227

Supplementary file Size Download File type/resource
GSM5747749_Tbx3_LB_E10_ChIP_rep2.bb 969.8 Kb (ftp)(http) BB
GSM5747749_Tbx3_LB_E10_ChIP_rep2.bw 603.3 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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