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Status |
Public on Dec 31, 2023 |
Title |
ChIP Tbx3 FL bud E9.75-E10.25 replicate 2 |
Sample type |
SRA |
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Source name |
forelimb buds
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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)
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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.
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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.
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina NextSeq 500 |
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Description |
For Tbx3 ChIP-seq, a 3XFlag epitope tag was inserted into the carboxy-terminal end of the endogenous Tbx3 coding region.
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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.
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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 |
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Relations |
BioSample |
SAMN24300609 |
SRA |
SRX13488227 |