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| Status |
Public on Aug 21, 2015 |
| Title |
6h J4 |
| Sample type |
SRA |
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| Source name |
multiple myeloma
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| Organism |
Homo sapiens |
| Characteristics |
cell line: myeloma cell line JJN-3
treatment: GSK-J4
time: 6h
chip antibody: anti-H3K4me3 (Millipore, catalog# 07-473, lot# 2207275)
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| Treatment protocol |
Cells were seeded the day before treatment. The final DMSO concentration for compound treatments and controls was 0.1 %. 5µM GSK-J4 or 5µM LNA was used for treatment. Compound treatments were carried out for 6h or 48h. LNA treatments were carried out for 7 days.
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| Growth protocol |
JJN-3 cells were maintained in 1:1 IMDM (Iscoves Modified Dulbeccos Medium, Lonza): DMEM (Dulbeccos Modified Eagle’s Medium, Sigma) with 20% FCS (foetal calf serum, Sigma) and 2 mM L-glutamine (Sigma). Cultured at 37oC in humidified cabinet at 5% CO2 (Heraeus Function Line).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed in 1% formaldehyde solution for 8 min followed by quenching of the reaction with glycine (125 mM) for 5 min. Cell were lysed in ChIP Lysis Buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl pH 8.1) and sonicated using Bioruptor Pico (Diagenode). Sonication was performed at 4°C using 5 sets of 10 cycles (each consisting of 30s sonication, 30s refraction). Protein A Sepharose beads (Sigma, P9424) were washed and pre-blocked in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 167mM NaCl, 16.7 mM Tris-HCl pH 8.1) with 5mg/ml BSA and 0.2mg/ml yeast tRNA (Sigma, R5636) for 2 hours at 4°C. Once sonication was complete, samples were diluted 10 times with ChIP dilution buffer and incubated with pre-blocked beads for 1 hour. Beads were then removed and the pre-cleared samples were incubated overnight with 2µg/sample of anti-H3K4me3 (Millipore, 07-473) at 4°C. Beads were washed twice with ChIP Low Salt Buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 150mM NaCl, 20mM Tris-HCl pH 8.1), once with ChIP High Salt Buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 500mM NaCl, 20mM Tris-HCl pH 8.1), once with ChIP LiCl Buffer (0.25M LiCl, 1% NP40, 1% deoxycholate, 1mM EDTA, 10mM Tris-HCl pH 8.1) and twice with TE buffer (1mM EDTA, 10mM Tris-HCl pH 8.0). Chromatin was eluted with ChIP Elution buffer (1% SDS, 100mM NaHCO3, 250mM NaCl) and de-cross-linked at 65°C for at least 4 hours. Protein was digested by adding 0.7mg/ml Proteinase K and heating at 55°C for at least 1 hour. DNA was isolated using 1.8 volumes of AMPureXP beads (Beckman Coulter, A63881).
DNA libraries for Next-Generation Sequencing (NGS) were prepared using NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (NEB, E7370S) and NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 1) (NEB, E7335S). Samples were 4-plexed on each lane and 50 bp single-ended reads were obtained.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
6h treatment with GSK-J4
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| Data processing |
Image analysis and base calling were performed using RTA 1.17.21.3 and CASAVA 1.8.2
The quality of the reads was assessed with FastQC v0.9.2. Reads below quality scores of 25 (Illumina 1.5 ecoding) and any adapter or universal primer sequences were trimmed and only reads longer than 24bp were retained.
Reads were aligned with bowtie v2.1.0 using the “--sensitive” parameter.
Peaks were called using MACS v2.0.10.20131028 by pre-estimating the parameter “-extsize” with predictd and then defining the latter value as well as additional parameters “--keep-dup auto --nomodel”.
Differential enrichment was assessed with MACS2 bdgdiff by normalizing with sequencing depth and providing the additional parameters “-g 60 -l 120”.
Genome_build: hg19
Supplementary_files_format_and_content: bigWig
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| Submission date |
Aug 21, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Laurynas Pliuskys |
| Organization name |
University of Oxford
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| Department |
NDORMS
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| Lab |
Professor Udo Oppermann Lab
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| Street address |
Windmill Rd
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| City |
Oxford |
| ZIP/Postal code |
OX3 7LD |
| Country |
United Kingdom |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE60626 |
Activation of the Integrated Stress Response by a Jumonji Histone Demethylase Inhibitor Induces Pro-Apoptotic Metabolic Reprogramming in Multiple Myeloma |
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| Relations |
| BioSample |
SAMN03001981 |
| SRA |
SRX684733 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1483895_112.bw |
182.7 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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