|
| Status |
Public on Apr 02, 2019 |
| Title |
ATACseq-GRKO-GFP-GRtetra_DEX_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
3617-KOGR cells, stably integrated P481R GFP-GRtetra, Dex-treated, ATAC
|
| Organism |
Mus musculus |
| Characteristics |
cell line: 3617-KOGR
cell type: mouse adenocarcinoma
stably expressing: P481R GFP-GRtetra
treatment: Dexamethasone
concentration: 100 nM
time: 1 h
|
| Treatment protocol |
Mouse adenocarcinoma and MEF cells were grow in steroid-depleted medium (10% charcoal stripped FBS in DMEM) for 24 h, and the cells were subsequently left untreated or treated 100 nM of dexamethasone for 1 h in ChIP-seq and ATAC-seq, and for 2 h in RNA-seq, prior extraction.
|
| Growth protocol |
Mouse adenocarcinoma and MEF cells were grown in Dulbecco's Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 25 U/ml penicillin and 25 µg/ml streptomycin, and 2mM L-Glutamine, and kept at 37 °C in a humidified 95% air/ 5% CO2 incubator. Mouse adenocarcinoma cells were also supplemented with 5 µg/ml of tetracycline to supress GFP-GR expression.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For ChIP-seq, lysates were clarified from sonicated nuclei, and protein-DNA complexes were isolated with specified antibody. For ATAC-seq, 100 000 isolated nuclei were treated with 2.5 µl Nextera Tn5 Transposase from Nextera kit. For RNA-seq, RNA was extracted using the PureLink RNA mini kit.
Libraries were prepared for sequencing using standard Illumina protocols.
For ChIP-seq, Illumina TruSeq Chip Sample Prep Kit; for ATAC-seq, Illumina Nextera DNA Library Prep Kit; for RNA-seq, Illumina Stranded Total RNA.
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
RTA 2.4.11 was used for Basecalling and Bcl2fastq 2.17 was used for demultiplexing allowing 1 mismatch.
Trimmomatic 0.36 was used for adapter and quality control
ChIP- and ATAC-seq alignment was performed by Bowtie2 (2.1.0) with Bowtie2 –p 8 –x bowtie2_ref/genome_prefix –U read1.fastq –S result.sam.
RNA-seq alignment was performed by TopHat (2.0.8) with tophat -G annotation.gtf -o ./ -r 10 --mate-std-dev 200 -p 16 --librarytype fr-firststrand bowtie2_annotation/prefix read1.fastq read2.fastq
Peaks were called using HOMER program version 4.9 with findPeaks with following parameters; FDR<0.001, 4x fold enrichment over control, 4x fold enrichment over local background, and >75 tags per site. In addition, untreated GR ChIP sample was used to further filter out noise from GR samples using default parameters on getDifferentialPeaks.
Genome_build: mm10
Supplementary_files_format_and_content: bedGraph (HOMER makeUCSCfile command) from alignment file.
|
| |
|
| Submission date |
Dec 29, 2017 |
| Last update date |
Apr 03, 2019 |
| Contact name |
Ville Paakinaho |
| E-mail(s) |
villepaakinaho@gmail.com
|
| Organization name |
University of Eastern Finland
|
| Department |
School of Medicine
|
| Lab |
Biomedicine
|
| Street address |
Yliopistonranta 8
|
| City |
Kuopio |
| ZIP/Postal code |
70210 |
| Country |
Finland |
| |
|
| Platform ID |
GPL21103 |
| Series (1) |
| GSE108634 |
Glucocorticoid Receptor Quaternary Structure Drives Chromatin Occupancy and Transcriptional Outcome |
|
| Relations |
| BioSample |
SAMN08276946 |
| SRA |
SRX3523212 |
