|
| Status |
Public on Jun 02, 2016 |
| Title |
Fetal liver Knockout of alpha globin regulatory element R2, biological replicate 1 [ATAC-seq] |
| Sample type |
SRA |
| |
|
| Source name |
Erythroid cells
|
| Organism |
Mus musculus |
| Characteristics |
knockout of enhancer-like component: regulatory element R2
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 |
SAMN04526277 |
| SRA |
SRX1608858 |
