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Sample GSM2594186 Query DataSets for GSM2594186
Status Public on Jun 15, 2017
Title Deproteinized DNA, genome-wide ATAC transposition background
Sample type SRA
Source name Erythroid cells
Organism Homo sapiens
Characteristics tissue: blood
genes analysed: Genome-wide
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.
Library strategy ATAC-seq
Library source genomic
Library selection other
Instrument model Illumina NextSeq 500
Description Genome-wide library of Tn5 transposed fragments
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
Submission date May 01, 2017
Last update date May 15, 2019
Contact name Jim Hughes
Phone 1865222113
Organization name University of Oxford
Department MHU
Lab Genome Biology Group
Street address Weatherall Institute Of Molecular Me
City oxford
ZIP/Postal code OX3 9DS
Country United Kingdom
Platform ID GPL18573
Series (1)
GSE86393 Direct and tissue-specific assessment of the damaging potential of regulatory SNPs
BioSample SAMN06856747
SRA SRX2772271

Supplementary file Size Download File type/resource
GSM2594186_ATACBack_HumanEry.wig.gz 257.4 Mb (ftp)(http) WIG
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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