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| Status |
Public on May 15, 2016 |
| Title |
Elk1 D8 |
| Sample type |
SRA |
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| Source name |
Elk1 chromatin binding at day 8 of M9-induced reprogramming
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| Organism |
Mus musculus |
| Characteristics |
cell type: tdMEFs at day 8 of reprogramming
chip antibody: Elk1 antibody (Epitomics, 1277-1, lot GR106696-8)
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| Treatment protocol |
M9-induced neural reprogramming was performed in M9 medium (50% Neural basal, 50% DMEM/F12/Glutamax, 1xN2, 1xB27 without vitamin A, 0.075% BSA, 0.1 mM nonessential amino acids, CHIR99021 3 mM, LDN193189 100 nM, A83-01 0.5 mM, Hh-Ag1.5 0.5 mM, retinoic acid 1 mM, SMER28 10 mM, RG108 10 mM, Parnate 2 mM, bFGF 10 ng/ml) at 5% O2 and 5% CO2 incubator at 37°C.
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| Growth protocol |
tdMEFs were cultured in MEF medium (Dulbecco’s modified Eagle’s medium supplemented with 10 % fetal bovine serum, 0.1 mM nonessential amino acids, and 2 mM Glutamax) in 5 % CO2 and 20 % O2 at 37 °C.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells at indicated time points were cross-linked by 1% formaldehyde, quenched with 0.125 M glycine. The chromatin DNA was extracted and sheared into 200-400 bp, and immuoprecipitated using a EZ-ChIP kit (Millipore, 17-371), and indicated antibodies.
Barcoded ChIP-Seq libraries were made from 1 to 10 ng of chromatin immunoprecipitated DNA (ChIP-DNA) using the NuGen Ovation Ultralow V2 kit. 13 cycles were used for PCR amplification of the libraries. Amplified libraries were checked for adapter artifacts by Agilent Bioanalyzer using DNA high sensitivity reagents and chips (Agilent). All libraries were quantified by qPCR using an Illumina Library Quantification kit (KAPA Biosystems). Libraries were pooled in equimolar amounts and run on the HiSeq 2500 sequencer with a single read 50 cycle run (Illumina).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
base-calling was done using CASAVA 1.8.4.
1. Reads are trimmed using the fastq-mcf program
2. The filtered reads are analyzed using the Fastqc program
3. The reads are aligned to the genome mm10 using bowtie2
4. Samtools is used to keep only the reads that do not map uniquely to the genome (mapq score >= 30)
5, bam2bed is used to convert bam files into bed files
6. Normalized Read intensity: The genome is divided into 20bp bins. The tag density is defined as the number of tags that map to within 75bp of each genomic bin. Tag density was then normalized according to method: ENCODE best practices; Landt et al., Genome Research 2012; (http://genome.cshlp.org/content/22/9/1813.full)
7. Peak Calling by comparing the normalized intensity with background tags (Paige et al., Cell 2012; (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3462257/)). Enriched bins were merged.
Genome_build: mm10
Supplementary_files_format_and_content: Normalized intensity of the regions with at least one peak in any of the time points from Elk1 ChIP or Gli2 ChIP
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| Submission date |
Mar 07, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Sheng Ding |
| Organization name |
THE J DAVID GLADSTONE INSTITUTES
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| Department |
Gladstone Institute of Cardiovascular Disease
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| Street address |
1650 Owens Street
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| City |
SAN FRANCISCO |
| State/province |
California |
| ZIP/Postal code |
94158 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE78976 |
Genome-wide chromatin binding of Elk1 and Gli2 in chemical induced neural reprogramming |
| GSE78978 |
CHIP-seq analysis for chemical-induced neural stem cells from defined fibroblasts |
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| Relations |
| BioSample |
SAMN04537312 |
| SRA |
SRX1618616 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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