 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jul 15, 2024 |
| Title |
ChIPRx-RNAPII-unp-DOX-OHT-N2 |
| Sample type |
SRA |
| |
|
| Source name |
SH-EP NMYC-ER
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: SH-EP NMYC-ER
cell type: Neuroblastoma
cell cycle_synchronization: unsynchronized
dox-treatment (shrna-tf3c5): induced (shRNA expression)
4-oht treatment_(nmyc_expresion): induced (NMYC expression)
treatment with_inhibitor: not applicable
spike-in: murine cells
chip antibody: Pol2 (8WG16) (sc-56767; Santa Cruz)
|
| Treatment protocol |
The indcution of NMYC in the SH-EP NMYC-ER cells was achieved by adding 4-OHT (200 nM) for 4 hours. The expression of an shRNA targeting TF3C5 in the SHEP-NMYCER cells was induced with 1 µg/ml doxycycline for 48 hours. Where indicated, SH-EP NMYC-ER cells were treated with DRB (100 µM) for 2 hours.
For ChIP-Rx samples, an exogenous control (spike-in) was used. For that purpose, murine NHO2A or NIH3T3 cells were added at a 1:10 cell ratio to the samples during the cell lysis step.
|
| Growth protocol |
Human neuroblastoma derived SH-EP cells expressing an exogenous and 4-OHT inducible NMYC allele (SH-EP-NMYC-ER), were grown in RPMI-1640 medium supplemented with with 10% horse serum and 1% penicillin/ streptomycin. Cells were cultured at 37C at 5% CO2.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For Cut&Run sequencing, cells were harvested with Accutase, washed and then coupled to ConA-coated magentic beads (Polysciences Europe). Cells were then permeabilized and incubated with the primary antibody (EXOSC5 #NBP2-14952; 1:100 diluted; Novus Biologicals) in antibody binding buffer over night at 4°C. Cells were then washed in the presence of digitonin and 700 ng/ml MNase was added per sample for 1 hour at 4°C.
For Cut&Run sequencing samples, the MNase treated cells were washed with low-salt rinse buffer. Incubation buffer, containing CaCl2 and digitonin, was added for 30 min at 4°C. The Mnase activity was stoped in the presence of 20 µg/ml Rnase A. DNA fragments were released at 37°C for 30 min and the decrosslinking was done for 1 hour at 50°C by adding 0.01% SDS and 10 mg/ml Proteinase K. DNA was extracted wit phenol-chloroform and resuspended in TE buffer.
For ChIP-seq and ChIP-Rx sequencing, cells were fixed for 5 min at RT with 1% formaldehyde and harvested in cold PBS contianing protease and phosphatase inhibitors. For ChIP-Rx samples, murine NHO2A or NIH3T3 cells were added at a 1:10 cell ratio during cell lysis to serve as an exogenous control (spike-in). Cell lysis was done for 20 min in PIPES buffer containing 85 mM KCl and 0.5% NP-40, followed by collection of nuclei by centrifugation.
For ChIP-seq and ChIP-Rx sequencing, the crosslinked chromatin was extracted from the nuclei in TRIS buffer with 150 mMCaCl, 1 mM EDTA and 1% NP-40, 1% sodium deoxycholate and 0.1% SDS. The chromatin was fragmented using the Covaris Focused Ultrasonicator M220 for 50 min. Dynabeads Protein A and Protein G were pre-incubated overnight with 10-15 µg antibody in the presence of 5 mg/ml BSA.
For ChIP-seq and ChIP-Rx sequencing, chromatin was added to the pre-incubated beads for at least 6h at 4°C with rotation. Beads were then washed with different TRIS buffers containing distinct salt-concentrations, EDTA and detergents, followed by wahsing with only TE-buffer. Chromatin was eluted with 100mM NaHCO3 and 1% SDS for 15 min. Protein and RNA were digested with proteinase K and RNase A. DNA was isolated by phenol-chloroform extraction followed by an ethanol precipitation. DNA was quantified using the Quant-IT PicoGreen dsDNA assay.
The DNA libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer’s instructions. Pre-PCR samples were purified and/or size-selected using AMPureXP beads. The libraries were PCR-amplified with the NEBNext Multiplex Oligos for Illumina Kit and again purified using Agencourt AMPure XP beads (Beckman Coulter). Library quality and fragment size distribution was analyzed on a Fragment Analyzer (Advanced Analytical). Sequencing was performed in single-read or paired-end mode on a NextSeq500 or NextSeq2000 platform (Illumina).
ChIP, ChIP-Rx and CUT&RUN followed by sequencing.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
| |
|
| Description |
ChIP-Rx-seq
|
| Data processing |
Basecalls were performed using bcl2fastq v1.1.0 (Illumina) and the quality of the generated FASTQ files was analysed with FastQC.
For Cut&Run sequencing, ChIP-seq and ChIP-Rx sequencing, reads were aligned to the human genome (hg19/GRCh37) using Bowtie2 v2.3.5.1 with default parameters. Normalization by aligned read number was performed on the input and on all correspondent IP samples. Where applicable, the number of spike-in normalized reads per sample was calculated by dividing the number of reads mapping exclusively to hg19 by the number of reads mapping exclusively to mm10 and multiplying this number with the smallest number of reads mapping to mm10 of all samples.
Following read-normalization, files were converted to bedGraph with bedtools genomecov v2.26 and visualized in a genome browser using the package plotgardener v1.012 in R v4.1.1. MACS2 v2.1.2 was used for peak calling for MYCN, TF3C5 and RAD21. H3K4me1, H3K4me3 and H3K27ac peaks were called with SICER v1.1.
Assembly: hg19
Supplementary files format and content: For CUT&RUN-seq, ChIP-seq and ChIP-Rx sequencing, the provided processed files are bedGraph files for data display in a genome browser.
|
| |
|
| Submission date |
Jan 17, 2023 |
| Last update date |
Jul 15, 2024 |
| 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 |
GPL30173 |
| Series (2) |
| GSE223052 |
TFIIIC and MYCN link the three-dimensional chromatin structure of promoters to transcription termination of stalled RNA polymerase (ChIPseq CnR) |
| GSE223058 |
TFIIIC and MYCN link the three-dimensional chromatin structure of promoters to transcription termination of stalled RNA polymerase |
|
| Relations |
| BioSample |
SAMN32769658 |
| SRA |
SRX19051717 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6938968_ChIPRx-RNAPII-unp-DOX-OHT-N2.bedGraph.gz |
123.9 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
