|
| Status |
Public on Oct 15, 2018 |
| Title |
shPU.1_kd_72h_PP |
| Sample type |
SRA |
| |
|
| Source name |
acute monocytic leukemia - genetically modified
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: acute monocytic leukemia - genetically modified
4c-seq viewpoint: PU.1 Promoter
|
| Treatment protocol |
N/A
|
| Growth protocol |
Inducible shRNA knockdown: 293TCeB cells were used for retroviral supernatant production after transfection with the shRNA-construct pRSMX_PG_along with the helper plasmids pHit/60 and pHIT/EA6x3 (PMID: 16572121, 11910072 and 12776180). Hereby, two tetracycline repressor (TETR) binding sites (Tet operators) were inserted into the histone H1 promoter for the inducible expression of shRNAs (TETON system). GFP+ selection of construct-positive cells was monitored by FACS analysis. Addition of 20 µg/ml doxycycline (Dox) activated transcription of the shRNA sequences.
Primary human monocytes: Blood cells were collected from healthy donors in compliance with the Helsinki Declaration. Peripheral monocytes were separated by leukapheresis followed by density gradient centrifugation with Ficoll/Hypaque purification and subsequent countercurrent centrifugal elutriation as previously described (PMID: 8864140). Monocytes were >85% pure as determined by morphology and expression of CD14 (data not shown).
Human AML samples: Fresh AML blasts were collected at the University Hospital Muenster from bone marrow aspirates with high blast infiltration. Prior to freezing the cell suspensions, density gradient centrifugation was done using Ficoll-Paque to isolate mononuclear cells. Immediately before processing, cells were thawed and verified to have at least 80% viability. Use of the patient material was approved by the Ethics Committee of University Hospital Muenster (approval no. 2007-390-f-S).
THP-1 cell differentiation: The human monocytic AML cell line THP-1 was cultured in RPMI1640 (Thermo Fisher Scientific) medium supplemented with 10% fetal bovine serum (Superiour, Biochrom). Differentiation into macrophages was induced by treatment with phorbol 12-myristate 13-acetate (PMA, 10 nM final, Sigma-Aldrich) and Cholecalciferol (Vitamin D3, VD3, 100 nM final, Sigma-Aldrich). After incubation for the indicated time points, only adherent cells were harvested.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
4C-seq was performed as previously described (PMID: 22609568). For chromatin digestion, NlaIII (NEB) and Csp6I (Thermo Scientific) were used as first and second restriction endonucleases. Inverse PCR was performed to amplify specific viewpoints at the PU.1 promoter, the PU.1 URE or the TET2 gene as control. Single-end sequencing was performed using the Illumina HiSeq2500 platform.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Library strategy: 4C-seq
Base calling was performed with Illumina's CASAVA 1.7.
4C-seq reads were aligned against the human reference genome (hg19 /GRCh38.p9) using the Burrows-Wheeler Alignment Tool (BWA, v. 0.6.2; max. 2 mismatches, default settings otherwise).
Aligned reads were mapped to a virtual restriction fragment library based on the chosen restriction enzyme sequences with the R/Bioconductor package Basic4Cseq.
Fragment data was RPM-normalized and filtered: Only uniquely aligned reads on non-blind restriction fragments were kept for analysis.
Genome_build: hg19 (GRCh37.67)
Supplementary_files_format_and_content: *_near-cis.bed: bed files were created with the R/Bioconductor package Basic4Cseq and include running median RPM values per valid fragment in the chosen near-cis region for the experiment's viewpoint.
|
| |
|
| Submission date |
Feb 15, 2018 |
| Last update date |
Oct 28, 2021 |
| Contact name |
Frank Rosenbauer |
| E-mail(s) |
[email protected]
|
| Organization name |
University Hospital Münster
|
| Department |
Institute of Molecular Tumor Biology
|
| Street address |
Robert-Koch-Str. 43
|
| City |
Münster |
| ZIP/Postal code |
48149 |
| Country |
Germany |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE110683 |
Temporal auto-regulation during PU.1 locus SubTAD formation (4C-seq) |
| GSE120143 |
Temporal auto-regulation during PU.1 locus SubTAD formation |
|
| Relations |
| BioSample |
SAMN08543157 |
| SRA |
SRX3703053 |
