|
| Status |
Public on Jan 08, 2021 |
| Title |
HEK293flpGR3KR_GR_DMSO-Dex_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
Isogenic HEK293 cells, SUMOylation-defective GR (GR3KR), dex, GR ChIP
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293
cell type: human embryonal kidney cell line
stably expressing: SUMOylation-defective GR (GR3KR)
treatment: DMSO + dexamethasone
concentration: 0.01% + 100 nM
time: 24 h + 1 h
chip antibody: GR (Santa Cruz, sc-1003, lot # D1812)
|
| Treatment protocol |
HEK293flpGR cells were seeded at 70 % confluence in 10-cm plates and allowed to grow in steroid-depleted medium (2.5 % charcoal stripped FBS in DMEM) 72 h and the cells were subsequently treated wither with vehicle (EtOH) or 100 nM of dexamethasone for 1 h prior ChIP or ATAC. For RNA-seq 6 h hormone treatment was used. SAE inhibitor treatment was 1 uM for 24 h prior extraction. HEK293flpPIAS1 cells were treated with 100 ng/ml of tetracycline for 24 h prior ChIP.
|
| Growth protocol |
HEK293flpGR 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 100 µg hygromycin-B, and kept at 37 °C in a humidified 95% air/ 5% CO2 incubator. HEK293flpPIAS1 cells were also supplemented with 15 µg/ml of blasticidin.
|
| 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 Qiagen RNeasy Mini kit.
For ChIP-seq, NEBNext Ultra II DNA Library Prep Kit; for ATAC-seq, Illumina Nextera DNA Library Prep Kit; for RNA-seq, NEBNext Poly(A) mRNA Magnetic Isolation Module kit and NEBNext Ultra II Directional RNA Library Prep kit.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Basecalls performed using CASAVA
FASTX-toolkit was used to trim low quality reads and sequence duplicates were collapsed
The reads were aligned to human reference genome version hg19 by using Bowtie software version 0.12.9
Peaks were called using HOMER program version 4.9. with default parameters in findPeaks. Rabbit IgG sample from HEK293flpFRT cells or mouse IgG sample from HEK293flpGR cells was used as control.
Alignment of RNA-seq data to human reference genome version hg19 was done with STAR. Differential expression of genes was analyzed using DESeq2 using HOMER.
Genome_build: hg19 (GRCh37)
Supplementary_files_format_and_content: bedGraph (HOMER makeUCSCfile command) from alignment file.
|
| |
|
| Submission date |
Dec 03, 2020 |
| Last update date |
Jan 10, 2021 |
| Contact name |
Ville Paakinaho |
| E-mail(s) |
[email protected]
|
| 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 |
GPL18573 |
| Series (2) |
| GSE64301 |
SUMOylation regulates the protein network and chromatin accessibility at glucocorticoid receptor-binding sites [sequencing] |
| GSE64373 |
SUMOylation regulates the protein network and chromatin accessibility at glucocorticoid receptor-binding sites |
|
| Relations |
| BioSample |
SAMN16989138 |
| SRA |
SRX9628173 |
