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| Status |
Public on Jul 15, 2024 |
| Title |
spLHiChIP-RAD21-OHT-DRB |
| Sample type |
SRA |
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| Source name |
SH-EP NMYC-ER
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| Organism |
Homo sapiens |
| Characteristics |
cell line: SH-EP NMYC-ER
cell type: Neuroblastoma
cell cycle_synchronization: unsynchronized
dox-treatment (shrna-tf3c5): not applicable
4-oht treatment_(nmyc_expresion): induced (NMYC expression)
treatment with_inhibitor: DRB (Benzimidazole; RNAPII CTD kinase inhibitor)
spike-in: murine cells
chip antibody: RAD21 (A300-080A; Bethyl Laboratories)
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| 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 MLN8237 (1 µM) or DRB (100 µM) for 2 hours.
For spike-in phosphorylated linker HiChIP (spLHiChIP) samples, an exogenous control (spike-in) was used. For that purpose, murine NHO2A cells were added at a 1:20 cell ratio to the samples during the nuclei isolation step.
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| 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.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For phosphorylated linker Hi-C (pLHi-C), phosphorylated linker HiChIP (pLHiChIP) and spike-in phosphorylated linker HiChIP (spLHiChIP), cells were cross-linked with 1% formaldehyde for 5 min and stopped by adding 125 mM glycine for 10 min at room temperature. Nuclei were isolated and a one-step in situ digestion and dephosphorylation took place by using DpnII and rSAP enzymes. During the nuclei isolation step, only for the spLHiChIP samples, murine NHO2A cells were added as exogenous control (spike-in) at a 1:20 cell ratio. Next, biotinylated oligos with 5’ end phosphate were used with T4 ligase for the ligation step.
Chromatin fragmentation was performed using the Covaris Focused Ultrasonicator M220 for 30 min per ml lysate. Samples were de-crosslinked overnight and Protein and RNA were digested with proteinase K and RNase A. DNA was isolated using ChIP DNA Clean & Concentrator (Zymo Research). Biotin pull-down was performed with MyOne Streptavidin C1 beads (Thermo Fisher Scientific) according to manufacturer’s instructions.
Only for pLHiChIP and spLHiChIP samples, the chromatin fragmentation was followed by an immunoprecipitation of the target protein. To this end, Dynabeads Protein A and Protein G were pre-incubated overnight with 10-15 µg antibody in the presence of 5 mg/ml BSA. Next, 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.
The immunoprecipitated chromatin of the pLHiChIP and spLHiChIP samples was eluted with 100mM NaHCO3 and 1% SDS for 15 min. Eluted samples were de-crosslinked overnight. DNA was isolated using ChIP DNA Clean & Concentrator (Zymo Research). Biotin pull-down was performed with MyOne Streptavidin C1 beads (Thermo Fisher Scientific) according to manufacturer’s instructions.
Library preparation was the same for all samples and was done on beads using the NEBNext ChIP-Sequencing Library Prep Master Mix Set for Illumina (NEB) according to manufacturer's instrcutions. The only deviation from the instructions was an additional washing step between each module using a Tris-buffer with 0.5 mM EDTA, 1 M NaCl and 0.05% Tween followed by a washing step with only TE buffer. 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).
pLHi-C sequencing (Illumina NextSeq500) and (s)pLHiChIP sequencing (Illumina NextSeq500 or NextSeq2000)
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
spLHiChIP-seq
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| Data processing |
Basecalls were performed using bcl2fastq v1.1.0 (Illumina) and the quality of the generated FASTQ files was analysed with FastQC.
For pLHi-C, pLHiChIP and spLHiChIP sequencing reads, the program HiC-Pro v2.11.4 was used to trim linker sequences, align to hg19, filter by DpnII restriction fragments and carry out qualtiy controls. HiC-Pro was also used to generate valid pairs outputs (bedpe files). For pLHi-C, the program HiC-Pro was further used to build contact maps and to transform those into HIC format (hic files).
For spLHiChIP, a spike-in (mouse; mm10) normalization was done. 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. For spLHiChIP and pLHiChIP valid pairs, DNA loops were called using the program hichipper v0.7.7.
Assembly: hg19 (GRCh37) and mm10
Supplementary files format and content: For pLHi-C, the provided processed files are hic files for display and further analysis of contact maps. For pLHiChIP and spLHiChIP sequencing experiments, the provided processed files are bedpe files used to describe pairs of genomic regions.
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| Submission date |
Jan 17, 2023 |
| Last update date |
Jul 15, 2024 |
| Contact name |
Martin Eilers |
| Organization name |
University of Wuerzburg
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| Department |
Chair for Biochemistry and Molecular Biology
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| Lab |
Martin Eilers
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| Street address |
Am Hubland
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| City |
Wuerzburg |
| ZIP/Postal code |
97074 |
| Country |
Germany |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE223054 |
TFIIIC and MYCN link the three-dimensional chromatin structure of promoters to transcription termination of stalled RNA polymerase (HiC HiChIP) |
| GSE223058 |
TFIIIC and MYCN link the three-dimensional chromatin structure of promoters to transcription termination of stalled RNA polymerase |
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| Relations |
| BioSample |
SAMN32769637 |
| SRA |
SRX19051930 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6939000_spLHiChIP-RAD21-OHT-DRB.bedpe.gz |
39.6 Kb |
(ftp)(http) |
BEDPE |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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