|
| Status |
Public on Oct 29, 2020 |
| Title |
Hypoxia_rep2_tech2 |
| Sample type |
SRA |
| |
|
| Source name |
MCF7
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF7
cell type: breast cancer
treatment: Hypoxia
|
| Growth protocol |
MCF-7 were cultured in DMEM low glucose medium (1g/l) supplemented with 10% FBS no longer than 20 passages. Cells were subjected to normoxia or 0.1% hypoxia for 48 hours using an InVivO2 chamber (Baker).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Assay for transposition of active chromatin sequencing (ATAC-Seq) was performed as previously published (Buenrostro, 2013) on single cell suspensions. 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). The libraries were quantified using the universal library quantification kit (KAPA Biosystems).
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Genome-wide library of transposed fragments
|
| Data processing |
Library Strategy: ATAC-seq
Mapping : bowtie -m 2 --maxIns 350, hg19
For un-mapping reads : trimming adaptors trim_galore -- length 10, combining overlapping R1-R2 reads : flash -m 9 -x 0.125
Combining trimmed+flashed and originally mapped reads, mapping again with bowtie -m 2 --maxIns 350
Removing read pairs overlapping Blacklisted regions
Removing duplicates : samtools rmdup
Reconstructing sequenced fragments from R1+R2 read pairs ( bedtools bedpe | cut -f 1,2,6) - for visualisation
Pileup of fragments (bedtools genomecov -counts)
Generating bigwig tracks (ucsctools bedGraphToBigWig)
Genome_build: hg19
Supplementary_files_format_and_content: bigWig : DEEPtools bamCoverage converison of filtered fragments (reconstructed, duplicate-filtered fragments : see above)from bam to bigwig, (--binSize 10 --normalizeUsingRPKM --minMappingQuality 30)
|
| |
|
| Submission date |
Jun 26, 2019 |
| Last update date |
Oct 29, 2020 |
| Contact name |
Damien Downes |
| E-mail(s) |
[email protected]
|
| Phone |
01865222374
|
| Organization name |
The University of Oxford
|
| Department |
MRC Weatherall Institute of Medicine
|
| Street address |
John Radcliffe Hospital
|
| City |
Headington |
| State/province |
Oxfordshire |
| ZIP/Postal code |
OX3 9DU |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL18573 |
| Series (1) |
| GSE133327 |
Chromatin response to hypoxia in MCF7 cells |
|
| Relations |
| BioSample |
SAMN12139320 |
| SRA |
SRX6367494 |
