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Status |
Public on Mar 16, 2012 |
Title |
E10.5_hindlimb_ChIP_input_rep4_index4 |
Sample type |
SRA |
|
|
Source name |
embryonic hindlimb bud, ChIP input
|
Organism |
Mus musculus |
Characteristics |
strain: C57BL/6J tissue: hindlimb bud developmental stage: gestational day E10.5 chip antibody: none sequencing run type: Paired-end 75 multiplexed
|
Treatment protocol |
Pregnant mothers were euthanized, and embryos were removed and placed in cold PBS. Forelimb and hindlimb buds were individually removed with forceps.
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Growth protocol |
Normal mouse gestation to day 10.5.
|
Extracted molecule |
genomic DNA |
Extraction protocol |
For each ChIP-Seq, limb bud tissue was crosslinked with 1% formaldehyde at room temperature and stored at -80°C. Chromatin was extracted and sheared by sonication. Soluble chromatin was combined with Protein G Dynabeads prebound with appropriate antibodies at 4C overnight. Beads were collected with magnet and washed 5x. Chromatin was eluted with TE+1%SDS at 65C for 10 minutes. Crosslinks were reversed at 65C overnight, and then chromatin was purified with the PCR cleanup kit (Qiagen).
Standard procedure included with the Illumina ChIP-Seq kit (IP-102-1001). For multiplexed samples, Illumina Multiplexing adapters and primers (PE-400-1001) were used instead of those provided with the ChIP-Seq kit. Multiplexed samples were paired-end sequenced with an insert size of 300 bp. Standard procedure included with the Illumina mRNA-seq kit (RS-100-0801).
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina HiSeq 2000 |
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|
Description |
Input
|
Data processing |
ChIP-Seq alignments: ChiP-Seq reads were aligned with bowtie (0.12.3) to the mouse genome (mm9) retaining only uniquely mapped reads (command: bowtie -m 1 -p 8 -B 1 --solexa1.3-quals ~/GENOME/mm9/dna/mm9_nh).
ChIP-Seq peak calling: ChIP peaks were identified by MACS (1.37) against input controls. Default parameters were used for CTCF ChIP-Seq (input control replicates 1 and 2)(command: macs -t ../../s_6_sequence.aligned -c ../../s_5_sequence.aligned --name=Mouse_CTCF_091310_FL_mfold10_p00001 --format=BOWTIE --tsize=74 --gsize=1860000000 --bw=150 -- pvalue=0.00001 --diag --mfold=10 --wig). Nomodel option was selected for H3K27me3 and H3K27ac ChIP seq (input control replicates 3 and 4 respectively)(command: macs -t ../082310/bowtie/s_5_sequence.aligned -c ../082210/bowtie/s_4_sequence.aligned --name=Mouse_h3k27me3_FL1_p00001 --nomodel --shiftsize=1 --format=BOWTIE --tsi ze=74 --gsize=1860000000 --bw=150 --pvalue=0.00001 --diag).
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Submission date |
Jul 13, 2011 |
Last update date |
May 15, 2019 |
Contact name |
Justin Cotney |
E-mail(s) |
cotney@uchc.edu
|
Organization name |
UConn Health
|
Department |
Genetics and Genome Sciences
|
Lab |
Cotney Lab
|
Street address |
400 Farmington Ave.
|
City |
Farmington |
State/province |
CT |
ZIP/Postal code |
06030-6403 |
Country |
USA |
|
|
Platform ID |
GPL13112 |
Series (1) |
GSE30641 |
Chromatin state signatures associated with tissue-specific gene expression and enhancer activity in the embryonic limb. |
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Relations |
SRA |
SRX083272 |
BioSample |
SAMN00668311 |
Named Annotation |
GSM759873_E10.5_hindlimb_ChIP_input_rep4.bw |