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Status |
Public on Nov 16, 2017 |
Title |
NRAS metastatic melanoma RNA-seq JQ1 (+) 24 hours Rep 2 [SKmel147] |
Sample type |
SRA |
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Source name |
Cell culture
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Organism |
Homo sapiens |
Characteristics |
cell line: SKmel147 passage: N/A
|
Treatment protocol |
treated with 10nM JQ1 (JQ1(+)) for 24 hours
|
Growth protocol |
grown in DMEM supplemented with 10% FBS, penicillin and streptomycin.
|
Extracted molecule |
total RNA |
Extraction protocol |
RNA was extracted using QIAGEN miRNeasy minikit. RNA was then processed with Ribo-Zero rRNA Removal Kit (Illumina) to remove rRNA. Totla RNA was processed into sequencing libraries using the Illumina ScriptSeq Complete Gold kit following manufacturer’s protocol.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2000 |
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|
Description |
RNA-seq JQ1 (+) 24 hours Rep 2 SKmel147-RNA-JQ1-gene_exp.diff RNA-gene_exp.diff
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Data processing |
Native ChIP-seq: Reads were trimmed from Illumina adapters using in-house script. Reads were aligned using Bowtie (version 0.12.7) with parameters –l 40-65 –n 2 –S –best –k 1 –m 20. BAM files were generated using SAMtools. bigwig files were generated from BAM files using deepTools bamCoverage (version 2.4.1) with parameters –normalizeUsingRPKM –binsize 10 XL ChIP-seq: Reads were trimmed from Illumina adapters using in-house script. Reads were aligned using Bowtie (version 0.12.7) with parameters -k 1 -m 1 --best -S -n 2 -l 65 -q. BAM files were generated using SAMtools. duplicated reads were removed using SAMtools. bigwig files were generated from BAM files using deepTools bamCoverage (version 2.4.1) with parameters –normalizeUsingRPKM –binsize 10. Significant peaks were called using MACS2 callpeak (version 2.1.1.2) with parameters -f BAM -g 2.7e9 -s 100 --bw 200 —slocal 1000 -q 0.01. ATAC-seq: Reads were trimmed from Illumina adapters using in-house script. Reads (40bp Paired-end) were aligned using Bowtie2 (version 2.1.0) with parameters -q -X 2000. BAM files were generated using SAMtools. Reads that align to mtDNA, with quality value Q<30, as well as duplicated reads, were discarded using in-house scripts and Samtools.. bigwig files were generated from BAM files using deepTools bamCoverage (version 2.4.1) with parameters –normalizeUsingRPKM –binsize 10. Significant peaks were called using MACS2 callpeak (version 2.1.1.2) with parameters –nomodel –nolambada –keepdup all –slocal 10000 RNA-seq: Reads (50bp, Paired-end) were aligned using TopHat (version 2.1.0). Transcriptome assemblies in FPKM, and differential expression ratios were computed with Cufflinks (version 2.1.1). In all samples, genes were called ‘expressed’ if normalized FPKM≥1.5. Genes were called downregulated upon JQ1 treatment if nFPKM≥1.5; log2FoldChange≤-0.625; and Padj<0.05. Genes were called overexpressed over NHM if nFPKM≥1.5 (in the melanoma sample); log2FoldChange≥2; and Padj<0.05. Genes were called downregulated upon AMIGO2 depletion if nFPKM≥1.5 (prior to depletion); log2FoldChange≤-1.2; and Padj<0.05. log2FoldChange≥1.2 for upregulated genes. SEs calling: Enhancers (TEs) and super enhancers (SEs) were called based on ChIP-seq BRD4 enrichment in SKmel147 using the ROSE (Rank Ordering of Super-Enhancers) algorithm (stitching distance 12.5Kb and TSS exclusion zone size 2.5Kb. BRD4 levels were normalized to Input control; ChIP-Input) Genome_build: GRCh37/hg19 Supplementary_files_format_and_content: fastq - raw illumina reads. bigwig - aligned reads pileup. diff - gene expression in normalized FPKM
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Submission date |
Feb 03, 2017 |
Last update date |
May 15, 2019 |
Contact name |
Emily Bernstein |
E-mail(s) |
emily.bernstein@mssm.edu
|
Phone |
(212) 824-9335
|
Organization name |
Mount Sinai School of Medicine
|
Department |
Oncological Sciences
|
Lab |
Bernstein Lab
|
Street address |
1470 Madison Avenue, 6th floor Rm 302
|
City |
New York |
State/province |
NY |
ZIP/Postal code |
10029 |
Country |
USA |
|
|
Platform ID |
GPL11154 |
Series (1) |
GSE94488 |
Harnessing BET inhibitor sensitivity reveals AMIGO2 as a melanoma survival gene. |
|
Relations |
BioSample |
SAMN06294919 |
SRA |
SRX2537535 |