GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM2842799

Query DataSets for GSM2842799

Status

Public on Nov 16, 2017

Title

Normal Human Melanocytes Input-XL [NHM1]

Sample type

SRA

Source name

Foreskin

Organism

Homo sapiens

Characteristics

cell type: foreskin, NHM1
passage: 8

Treatment protocol

untreated

Growth protocol

cultured in Melanocyte Growth Media 254 supplemented with Human Melanocyte Growth Supplement (Life Technologies), CaCl2 (0.2uM, Life Technologies: 50-9702) and PMA (10 ng/ml, Sigma: P8139).

Extracted molecule

genomic DNA

Extraction protocol

For FOSL2 and TEAD4, 10x10^6 cells, were crosslinked with 0.25M DSG for 45min, followed by 1% PFA crosslinking for 10 min at RT. ChIP was performed essentially as previously described (Tian et al., Two-step Crosslinking for Analysis of Protein-chroamtin Interactions. Methods Mol Biol, 2012. 809:105-120. Roe, J.S., et al., BET Bromodomain Inhibition Suppresses the Function of Hematopoietic Transcription Factors in Acute Myeloid Leukemia. Mol Cell, 2015. 58(6): p. 1028-39).
Libraries for ChIP-seq were done as previously described (Hasson D., et al., The octamer is the major form of CENP-A nucleosomes at human centromeres. Nat Struct Mol Biol. 2013 Jun;20(6):687-95).

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina NextSeq 500

Description

Input-XL

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

Nov 06, 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