|
| Status |
Public on Dec 20, 2017 |
| Title |
SHEP_NMYCER_Sphase_arrested_RNAPII_N20_plusOHT_DMSO |
| Sample type |
SRA |
| |
|
| Source name |
SH-EP-NMYCER
|
| Organism |
Homo sapiens |
| Characteristics |
treatment 1: 4-OHT
treatment 2: DMSO
antibody: RNAPII (N20, Santa Cruz, sc-899)
|
| 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 |
genomic DNA |
| Extraction protocol |
Cells were treated with 1% formaldehyde for 10 min at 37 °C. After cell lysis, nuclei were re-suspended in RIPA buffer (10 mM Tris/HCl pH 7.5, 150 mM NaCl, 1% NP40, 1% deoxycholic acid (DOC), 0.1% SDS, 1 mM EDTA) and DNA was fragmented to a size <500 bp using a Branson sonifier. Chromatin was eluted with 1% SDS and crosslinking was reverted overnight. For purification, chloroform/phenol extraction was used.
Purified DNA was end-repaired, A-tailed, ligated to Illumina adaptors, size-selected (200 bp) and purified with Qiagen gel extraction kit. DNA fragments were amplified by 18 cycles of PCR and library size was tested with the Biorad Experion system. The amount of library DNA was quantified using a picogreen assay
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
For ChIP-seq, fastq files were mapped to the human genome (hg19) using Bowtie v1.1.1. and normalized to the sample with the smallest number of mapped reads.
Wiggle files were generated using MACS v1.4.2 with default parameters but a p-value cutoff of 1e-6 (N-MYC, TF3C5) and keep-dup parameter =1. .bedGraph files were generated using bedtools genomecov.
genome build: hg20
Fixed step wiggle files with a resolution of 10bp.
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 |
SAMN06831035 |
| SRA |
SRX2764659 |
