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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jun 15, 2017 |
| Title |
Primary erythroid cells, biological replicate 2, growth protocol 2 (human) |
| Sample type |
SRA |
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| Source name |
Erythroid cells
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| Organism |
Homo sapiens |
| Characteristics |
tissue: blood
genes analysed: Genome-wide
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| Treatment protocol |
Mouse: Phenylhydrazine (PHZ) injections, 4 in the course of 6 days.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Mouse: A single cell suspension was made by gently dissociating the PHZ spleens and passing it through a 70um filter. Cells were washed in cold PBS, and counted. 50million cells were used for the DNase-Seq protocol. Nuclei were isolated by lysing the cells and digested with increasing concentrations of DNaseI to isolate open chromatin fragments as previously published (Hosseini, 2014).
Human: Primary erythroid stem cell progenitors were isolated from peripheral blood, using CD34 coupled magnetic beads. Cells were expanded for 7days in low Epo conditions (0.5 IU/ml)) and then transfered for differentiation in high Epo (3.0 IU/ml) medium. On day 13, cells were washed in cold PBS, and counted. 50million cells were used for the DNase-Seq protocol. Nuclei were isolated by lysing the cells and digested with increasing concentrations of DNaseI to isolate open chromatin fragments as previously published (Hosseini, 2014, DOI 10.1371/journal.pgen.1003570).
The DNase-Seq protocol was perfromed as previously published (Hosseini 2014). DNA was purified using a phenol choroform extraction and the optimal disgests were selected for library preparation using NEB Library Preparation kit. Libraries were amplified and barcoded using the NEBNext 2xMastermix (NEB) and TruSeq oligos. A size selection step for small fragments was performed. DNase-Seq libraries profiles were visualized using D1000 tape on the Tapestation (Agilent). The libraries were quantified using the universal library quantification kit (KAPA Biosystems). Samples were sequenced on Illumina platform using: 150bp paired end reads (MiSeq), 75bp paired end reads (HiSeq) or 40/75 bp (NextSeq) paired end reads. For the background libraries, 100ng of genomic DNA was incubated with DNase for 3 minutes and then libraries were created to test sequence for sequence bias.
The strategy was to isolate open chromatin fragments specifically as they underly regulatory genomic regions. These are short stretches of DNA, with low abundance in the genome and thus require to be processed efficiently. The fragments can then be multiplexed and sequenced.
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| Library strategy |
DNase-Hypersensitivity |
| Library source |
genomic |
| Library selection |
DNAse |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Genome-wide library of DNase I digested fragments
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| Data processing |
Alignment to the genome Bowtie(1.1.2); default with -m set to 2
Trim_galore(0.3.1) (to remove sequencing adaptors from unmapped reads)
Flashing reads using flash(1.2.8); default with -m 9 -x 0.125 (to combine short overlapping, unmapped reads)
Realignment of formerunmapped reads using Bowtie as described before
Removal of PCR duplicates, using samtools(0.1.19)
Excluding regions highly affecting uniqueness and mappability using bedtools(2.17.0)
Merge bam files from same sample and same sequencing instrument model, keeping R1 and R2 separate.
Merge bam files from same sample for coverage estimation. Estimate coverage by aligned reads in a moving 300 bp window with a moving increment of 30 bp using custom perl script.
Genome_build: hg18/mm9
Supplementary_files_format_and_content: bigWig, window averaged read coverage
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| Submission date |
Sep 02, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Jim Hughes |
| E-mail(s) |
jim.hughes@imm.ox.ac.uk
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| Phone |
1865222113
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| Organization name |
University of Oxford
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| Department |
MHU
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| Lab |
Genome Biology Group
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| Street address |
Weatherall Institute Of Molecular Me
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| City |
oxford |
| ZIP/Postal code |
OX3 9DS |
| Country |
United Kingdom |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE86393 |
Direct and tissue-specific assessment of the damaging potential of regulatory SNPs |
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| Relations |
| BioSample |
SAMN05726806 |
| SRA |
SRX2105478 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2301522_CD34_D13_8_dnase_windowed_hg18.bedgraph.gz |
65.8 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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