|
| Status |
Public on Mar 19, 2015 |
| Title |
Mef2dIP_Mef2d_ChIP_WT_unstim_Ctx2 |
| Sample type |
SRA |
| |
|
| Source name |
retina
|
| Organism |
Mus musculus |
| Characteristics |
tissue: retina
genotype: wild type
age: postnatal day 12
chip antibody: anti-MEF2D (Flavell et al., 2008)
|
| Treatment protocol |
Retinae were dissected from p11 knockout or wild-type littermate controls.
|
| Growth protocol |
Mice were housed under standard conditions in the animal facility and dissected at the same time point in the light-dark cycle for each dissection.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
MEF2D ChIP from mouse retinas was performed as previously described for brain tissue (Hong et al., 2008) with the following modifications: p11 mouse retinas were dissected in ice-cold HBSS prior to homogenization and crosslinking. 4μg of anti-MEF2D antibody was pre-bound to 15μl of Protein A dynabeads (Life Technologies) per immunoprecipitation (IP) from approximately 100 million retinal cells. H3K27Ac ChIP was performed as described above with the following modifications: 10 mM sodium butyrate was added to all solutions until post-IP washes with the exception of cross-linking buffer. Chromatin was fragmented for H3K27Ac ChIP by brief sonication followed by MNase (New England Biolabs) digestion for 8 minutes at 37C to generate mononucleosomes. 0.25μg of anti-H3K27Ac antibody was used per IP from 10 million retinal cells. After reverse crosslinking all samples were purified using phenol/chloroform/isoamyl alcohol followed by column clean up.
Single-end short read sequencing libraires were prepared using standard protocols, following the manufacturers instructions. were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Base calling was performed using CASAVA software.
Illumina raw reads in .fastq files were aligned to mouse mm9 reference genome with bwa 0.6.2. Uniquely mapped reads were extended in silico to reflect average fragment size of the input DNA(~250bp).
Genome_build: mm9
Supplementary_files_format_and_content: bigWig files of unnormalized histograms for mapped, in silico extended reads
|
| |
|
| Submission date |
Sep 12, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Timothy J Cherry |
| Phone |
6174705859
|
| Organization name |
Seattle Children's Research Institute
|
| Department |
Developmental Biology and Regenerative Medicine
|
| Lab |
Cherry Lab
|
| Street address |
1900 9th Ave.
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98101 |
| Country |
USA |
| |
|
| Platform ID |
GPL13112 |
| Series (2) |
| GSE61391 |
MEF2D drives photoreceptor development through a genome-wide competition for tissue-specific enhancers (ChIP-Seq) |
| GSE61392 |
MEF2D drives photoreceptor development through a genome-wide competition for tissue-specific enhancers |
|
| Relations |
| BioSample |
SAMN03067474 |
| SRA |
SRX700326 |
