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Sample GSM2077312 Query DataSets for GSM2077312
Status Public on Jun 02, 2016
Title Fetal liver Knockout of alpha globin regulatory elements R2 and R3 biological replicate 3 [ATAC-seq]
Sample type SRA
 
Source name Erythroid cells
Organism Mus musculus
Characteristics knockout of enhancer-like component: regulatory element R2 and R3
strain: F1 C57Bl6 / CBA
tissue: Fetal Liver
age: E14.5
genes analysed: Genome-wide
Extracted molecule genomic DNA
Extraction protocol A single cell suspension was made by gently dissociating the E14.5 fetal livers and passing it through a 70um filter. Ter119 selection was performed by staining the cells with phycoerythrin conjugated anti-ter119 antibodies (BD biosciences)). The cells were subsequently washed and then incubated with MACS anti-phycoerythrin beads. Cells were then separated using a magnetic column, MACS (miltenyi biotec). Nuclei were isolated by lysing the cells as  previously published (Buenrostro, 2013).
Assay for transposition of active chromatin sequencing (ATAC-Seq) was performed as previously published (Buenrostro, 2013). 60000-80000 cells were used per biological replicate. Cells were lysed and nuclei were isolated prior to transposition with Tn5 transposase (Nextera, Illumina) for 30 minutes at 37°C. DNA was purified using a MinElute kit (Qiagen). Libraries were amplified and barcoded using the NEBNext 2xMastermix (NEB) and the custom primers as published in Buenrostro et al., 2013. ATAC-Seq libraries profiles were visualized using D1000 tape on the Tapestation (Agilent). Due to the broad range of DNA fragment sizes found in these libraries, quantitation with the Qubit for DNA concentration was found to be highly variable and was omitted. The libraries were quantified using the universal library quantification kit (KAPA Biosystems). Samples were sequenced using: 75bp paired end reads (MiSeq platform) or 40 bp paired end reads (NextSeq platform).
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 use of the transposase is ideal as the modified Tn5 can in one step identify the open chromatin fragments, cut them from the chromatin environment and ligate adaptors. The fragments can then be multiplexed and sequenced
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina NextSeq 500
 
Description Genome-wide library of transposed fragments
Data processing Library strategy: ATAC-seq
Trim_galore (to remove sequencing adaptors)
Alignment to the genome Bowtie 1 standard parameters with -m set to 2
Removal of PCR duplicates;
plot density of alignments in a moving 300bp window with an moving increment of 30 bp
Genome_build: mm9
Supplementary_files_format_and_content: wig
 
Submission date Mar 01, 2016
Last update date May 15, 2019
Contact name Jim Hughes
E-mail(s) jim.hughes@imm.ox.ac.uk
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 GPL19057
Series (2)
GSE78800 Dissection of a super-enhancer in vivo (ATAC-seq)
GSE78835 Genetic dissection of the α-globin super-enhancer
Relations
BioSample SAMN04526290
SRA SRX1608871

Supplementary file Size Download File type/resource
GSM2077312_R2R3_KOC.wig.gz 29.9 Mb (ftp)(http) WIG
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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