|
| Status |
Public on Dec 20, 2017 |
| Title |
SHEP_NMYCER_minusOHT_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
SH-EP-NMYCER
|
| Organism |
Homo sapiens |
| Characteristics |
treatment 1: EtOH
|
| Treatment protocol |
For siRNA transfections, cells were transfected using the RNAiMAX reagent and OptiMem medium (LifeTechnologies) according to the manufacturer's instructions. Cells were harvested 48 h after transfection. Cells were treated with CD532 (1µM) or DMSO for 4 (ChIP-seq) or 4 and 8 (RNA-seq) hours.To activate N-MYC-ER in SH-EP cells, cells were treated with 4-OHT (200nM) for 5 hours. Conditional shRNA-mediated depletion of TFIIIC5 was induced with doxycycline (1 µg/ml) for 48 hours.
|
| Growth protocol |
Neuroblastoma cells were grown in RPMI-1640 (Sigma). Medium was supplemented with 10% fetal calf serum (Biochrom) and penicillin/streptomycin (Sigma).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using RNeasy mini columns (Qiagen) including on-column DNase I digestion.
mRNA was extracted using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB). Library preparation was performed with the NEBNext® Ultra™ RNA Library Prep Kit for Illumina following the instruction manual. Size-selection of libraries was performed using Agencourt AMPure XP Beads (Beckman Coulter) followed by amplification with 12 PCR cycles. Afterwards libraries were quantified and the size was determinded using Experion Automated Electrophoresis System (Bio-Rad).
For RNA-seq, reads were mapped to hg19 with Tophat2 and Bowtie1 with default settings.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
SHEP_NMYCER_plusOHT_vs_minusOHT.txt
|
| Data processing |
Reads per gene were counted using the countOverlaps function from the R package {GenomicRanges}.
Weakly expressed genes were removed(mean count over all samples <1.5) and differentially expressed genes were called using EdgeR.
genome build: hg19
txt files containing Ensembl gene ID, gene symbol, gene description, log2FC, p-value and q-value (FDR) for all comparisons. A matrix file containing raw read counts for all samples.
Fastq files were generated using Illumina's CASAVA software and overall sequencing quality was analyzed with the FastQC script.
|
| |
|
| Submission date |
Apr 25, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Martin Eilers |
| Organization name |
University of Wuerzburg
|
| Department |
Chair for Biochemistry and Molecular Biology
|
| Lab |
Martin Eilers
|
| Street address |
Am Hubland
|
| City |
Wuerzburg |
| ZIP/Postal code |
97074 |
| Country |
Germany |
| |
|
| Platform ID |
GPL18573 |
| Series (1) |
| GSE78957 |
Association with Aurora-A controls N-MYC-dependent promoter escape and pause release of RNA polymerase II during the cell cycle |
|
| Relations |
| BioSample |
SAMN06831043 |
| SRA |
SRX2764651 |
