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Sample GSM2589150 Query DataSets for GSM2589150
Status Public on Dec 20, 2017
Title SHEP_NMYCER_Sphase_arrested_RNAPII_N20_plusOHT_CD532
Sample type SRA
 
Source name SH-EP-NMYCER
Organism Homo sapiens
Characteristics treatment 1: 4-OHT
treatment 2: CD532
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 SAMN06831033
SRA SRX2764661

Supplementary file Size Download File type/resource
GSM2589150_SHEP_NMYCER_Sphase_arrested_RNAPII_N20_plusOHT_CD532.bedgraph.gz 81.6 Mb (ftp)(http) BEDGRAPH
SRA Run SelectorHelp
Processed data provided as supplementary file
Raw data are available in SRA

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