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| Status |
Public on Mar 13, 2018 |
| Title |
Oct4 E75 LSEB 6 |
| Sample type |
SRA |
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| Source name |
Late Streak, Early Bud Embryo
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| Organism |
Mus musculus |
| Characteristics |
fusion protein: Oct4
cell type: Embryonic Day 7.5 (E75) - Late Streak, Early Bud (LSEB) Embryo
cell number: NA
replicate number: 6
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| Growth protocol |
Epiblast samples were dissected from 7.5 dpc embryos classified by morphological criteria.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted with the Quick-gDNA™ MicroPrep (ZymoResearch) kit according to the manufacturer's instruction.
Extracted gDNA was digested with DpnI (NEB, R0176S) and DamID adapter were then ligated using T4 DNA Ligase (M0202L). After ligation, the DNA was digested with DpnII (R0543S) and the DamID fragments of interest were amplified with the KAPA HiFi Polymerase (Roche, 07958935001). DNA was then purified using homemade SPRI magnetic beads and 50 ng of DNA were used as input for DamID-seq library preparation by using the Nextera® DNA Sample Preparation Kit (according to manufacturer's instructions). The libraries were quantitated using the The Qubit® 2.0 Fluorometer (Thermo Fisher). The DNA size distribution of the library was measured by an Agilent Bioanalyzer. Each sample was prepared in 3 or 4 biological replicates.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Library strategy: DamID-seq
Read quality was checked using FastQC (version 0.11.5) and adapters were trimmed using cutadapt (version 1.13).
Reads were aligned to the UCSC mm10 assembly of the mouse genome [3] using bwa mem [4] (version 0.7.15).
Unmapped, secondary, and supplementary alignments were filtered using SAMtools [5] (version 1.4).
Reads mapped to ENCODE blacklist regions [6], alternate, unplaced, and mitochondrial contigs were filtered using BEDtools [7] (version 2.26.0).
Reads were counted into fragments using the featureCounts command from Subread [8] (version 1.5.0).
Fragments with 10 read counts or lower in equivalent average log count-per-million (aveLogCPM) values were removed.
Normalisation factors were calculated with the normOffsets function from the csaw package (version 1.8.1). The type argument was set to “scaling”.
Using these normalisation factors, log count-per-million (logCPM) values were calculated and smooth quantile normalised using the qsmooth package (version 0.0.1)
Differentially bound fragments were identified by testing for differential abundance between the Dam-POI (Protein-of-Interest) and Dam-only samples. The normOffsets/qsmooth normalised count matrix was used with limma-trend from the limma package (3.30.13). The trend and robust arguments were set to TRUE.
Fragments were then combined into differentially-bound regions using the mergeWindows and combineTests function from the csaw package. The tol and max.width arguments were set to 260 (median GATC fragment size for the mm10 assembly) and 10,000, respectively.
Peaks were defined as regions with a false discovery rate (FDR) smaller than 0.1 and a log fold-change (logFC) greater than 0.5.
Read coverage across the genome was calculated with the genomecov command from BEDtools (version 2.26.0). Coverage for each sample was scaled using the normalisation factors calculated previously.
The bedGraph files were converted into bigWig files with the UCSC bedGraphToBigWig tool [11] (version 4). Subtracted bigWig files were generated using the diff command from WiggleTools [12] (version 1.2).
Genome_build: mm10
Supplementary_files_format_and_content: Peak calls are in narrowPeak file format
Supplementary_files_format_and_content: Read coverage is in bigWig file format
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| Submission date |
Apr 24, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Keisuke Kaji |
| E-mail(s) |
kkaji@exseed.ed.ac.uk
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| Organization name |
University of Edinburgh
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| Department |
MRC Centre for Reproductive Health
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| Street address |
47 Little France Crescent
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| City |
Edinburgh |
| State/province |
Midlothian |
| ZIP/Postal code |
EH16 4TJ |
| Country |
United Kingdom |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE98092 |
Mammalian low cell number and in vivo DamID-seq for transcription factor binding analyses |
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| Relations |
| BioSample |
SAMN06821067 |
| SRA |
SRX2754704 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2587030_Oct4_E75_LSEB_Pos_6.bigWig |
130.0 Mb |
(ftp)(http) |
BIGWIG |
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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