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| Status |
Public on Jun 15, 2017 |
| Title |
Deproteinized DNA, genome-wide ATAC transposition background |
| 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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| Extracted molecule |
genomic DNA |
| Extraction protocol |
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 10, cells were washed in cold PBS, and counted. 70,000 cells were used for the ATAC-seq protocol. Nuclei were isolated by lysing the cells and transposition with Tn5 transposase, DNA extraction and sequencing was performed as published by Buenostro et al 2013.
ATAC-seq was performed as previously published (Buenrostro:2013). Nuclei of 70000 lysed cells were isolated and transposition with Tn5 transposase (Nextera, Illumina) was performed for 30 minutes at 37 °C. The DNA was extracted using a MinElute kit (Qiagen). Libraries were then amplified and barcoded using the NEBNext 2xMastermix (NEB) and the custom ATAC-seq primers published by Buenrostro and colleagues. ATAC-seq libraries profiles were visualized using D1000 tape on the Tapestation (Agilent) and quantified using the universal library quantification kit (KAPA Biosystems). For the ATAC-seq background 100 ng of genomic DNA was incubated with the Tn5 transposase and the library was made following the protocol described before. 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 |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Genome-wide library of Tn5 transposed 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 former unmapped 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
Supplementary_files_format_and_content: wig, window averaged read coverage
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| Submission date |
May 01, 2017 |
| 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 |
SAMN06856747 |
| SRA |
SRX2772271 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2594186_ATACBack_HumanEry.wig.gz |
257.4 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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