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Sample GSM2476390

Query DataSets for GSM2476390

Status

Public on Nov 16, 2017

Title

NRAS metastatic melanoma RNA-seq shA2 #2 Rep 2 [SKmel147]

Sample type

SRA

Source name

Cell culture

Organism

Homo sapiens

Characteristics

cell line: SKmel147
passage: N/A

Treatment protocol

cells stably transduced with shA2 #2 lentiviral constructs

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.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

RNA-seq shA2 #2 Rep 2
SKmel147-RNA-AMIGO2KD-gene_exp.diff

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

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