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| Status |
Public on Oct 23, 2013 |
| Title |
MEF_H3.3_24h_r2 |
| Sample type |
SRA |
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| Source name |
NIH/3T3 Tet-On® 3G cells
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| Organism |
Mus musculus |
| Characteristics |
cell line: NIH/3T3 fibroblast cell line
treatment: aphidicolin
h3.3 induction (hour): 24h
chip antibody: HA
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| Treatment protocol |
MEFs were grown to 100% confluence and treated with 1ug/ml aphidicolin for 18 hours. Adding 3ug/ml doxycycline hyclate before crosslinking with formaldehyde at various time points induced HA/FLAG-H3.3 expression.
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| Growth protocol |
cDNA of human H3.3B or mouse H3.1 (Hist1h3h) was subcloned in-frame with a HA and FLAG tag at the C-terminus and cloned into the lentiviral plvx-Tight-Puro Vector (Clontech). Lentiviral particles were packaged in 293T cells with the psPAX2 packaging plasmid. Subsequently, we transduced NIH/3T3 Tet-On® 3G cells (Clontech) and drug-selected with puromycin for stable integration. NIH/3T3 MEFs were cultured in standard conditions with medium containing tetracycline-compatible FBS.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were crosslinked with formaldehyde treatment and chromatin fragmented to 200 to 300 bp by sonication. Chromatin from 2 × 107 cells was used for each ChIP experiment, which yielded approximately 200 ng of DNA. Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody.
Libraries were prepared according to Illumina's instructions. The ChIP DNA ends were repaired using PNK and Klenow enzyme, followed by treatment with Taq polymerase to generate a protruding 3′ A base used for adaptor ligation. Following ligation of a pair of Solexa adaptors to the repaired ends, the ChIP DNA was amplified using the adaptor primers for 17 cycles and the fragments around 220 bp (mononucleosome + adaptors) isolated from agarose gel. Libraries were sequenced on the Hi-seq following the manufacturer's protocols. As described in Barski et al., 2007 (A. Barski, S. Cuddapah, K. Cui, T.Y. Roh, D.E. Schones, Z. Wang, G. Wei, I. Chepelev and K. Zhao, High-resolution profiling of histone methylations in the human genome, Cell 129 (2007), pp. 823-837).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
ChIP-seq
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| Data processing |
Basecalls performed using GAPipeline 1.3.2 or CASAVA 1.0
ChIP-Seq reads were mapped to the mouse genome (mm9, or NCBI 37) using Bowtie, only allowing ‘one read per position’ matching. Redundant reads were removed from each ChIP-Seq library.
We identified ChIP-Seq tag enriched peaks using SICER. For calling of H3.3 peaks, we set the window size =200bp and gap size =600bp. Peaks with a false discovery rate (FDR) of higher than 0.01 were excluded from the analysis, compared with the input control libraries.
The total number of H3.3 peaks was identified by peak-calling at the 72h time point when both early-appearing and late-appearing peaks were readily detected. The number of reads in each peak region was recorded and normalized over the total mapped reads in the library. We obtained the H3.3/molecules relative enrichment of each peak by normalizing over input.
Genome_build: mm9
Supplementary_files_format_and_content: Bed: plan text file. Each line is one read. The six columns are chrom, start position, end position, length, alignment score and strand.
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| Submission date |
Oct 21, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Wenfei Jin |
| E-mail(s) |
[email protected]
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| Organization name |
NHLBI,NIH
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| Department |
Systems Biology Center
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| Lab |
Laboratory of Epigenome Biology
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| Street address |
9000 Rockville Pike
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE51505 |
Genome-wide incorporation dynamics reveal distinct categories of turnover for the histone variant H3.3 |
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| Relations |
| BioSample |
SAMN02380017 |
| SRA |
SRX366549 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1246669_GA4972_MEF_H3.3_24h.bed.gz |
127.2 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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