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Sample GSM2305254

Query DataSets for GSM2305254

Status

Public on Sep 08, 2016

Title

LNCaP_N-Myc_GSK343#1

Sample type

SRA

Source name

LNCaP cells

Organism

Homo sapiens

Characteristics

antibody: GSK343 (Sigma Aldrich SML0766) at 5uM for 6 days
tissue: LNCaP cells

Treatment protocol

cell lines were culture in RPMI supplemented with 10% of FBS (Life technology) and a cocktail of penicillin/streptomycin (Life technology).
EZH2 siRNAs were purchase from Dharmacon as On-target plus siRNA and transfected as a final concentration of 60nM with lipofectamine RNAimax (Life technology). Cells were treated with GSK343 (Sigma Aldrich SML0766) at 5uM for 6 days

Growth protocol

LNCaP and 22RV1 cell lines were culture in RPMI supplemented with 10% of FBS (Life technology) and a cocktail of penicillin/streptomycin (Life technology).

Extracted molecule

genomic DNA

Extraction protocol

For mouse tissues frozen embedded prostate H&E were assessed for pathology and normal, HGPIN or prostate cancer lesions were core from the frozen blocks and the RNA extracted using RNA simply Kit (Promega). For cell line, RNA extraction was performed using Trizol (Life technology) following manufacturer recommendation.
For the ChIp-Seq: Fifty million LNCaP-N-Myc or LNCaP control cells were washed in PBS twice and then fixed using 1% formaldehyde for 8 minutes at room temperature and quenched 5 min using 125 mM glycine. The cells were centrifuged and the cell pellet was resuspended in 2 milliliters of dilution buffer (10mM Tris HCL pH8.0, 0.1% SDS, 1.0mM EDTA with protease and phosphatase inhibitors). Protein-bound chromatin was fragmented by sonication for 15 minutes (Covaris series E220). H3K27me3 antibody was purchase from Diagenode (A1811-001P).
Libraries were prepared according to Illumina's instructions

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina Genome Analyzer

Data processing

Sequencing reads were aligned using STAR (Dobin et al., 2013) to hg19
HTSeq (Anders et al., 2015) and Cufflinks (Trapnell et al., 2012) were then used to generate read counts and FPKM respectively
For the LNCaP samples, the gene counts were used with DESeq2 (Love et al., 2014) to identify differentially expressed genes, defined to be those with adjusted p < 0.10 and absolute log2 fold change > 1
For the mouse samples, Cuffdiff (Trapnell et al., 2012) was then used to identify differentially expressed genes, defined to be those with p < 0.05.
For the H3K27me3 ChIP-seq: Sequencing reads were aligned to hg19 using the Burrows-Wheeler Alignment (bwa) Tool (Li and Durbin, 2009). Genes were considered direct targets if there was more read coverage by H3K27me3 than input at promoter regions, which were defined to be 2kb upstream of the transcription start site of RefSeq transcripts. Coverage plots were generated using the Integrative Genomics Viewer (IGV) (Robinson et al., 2011). H3K27me3 levels were normalized by input and differences in log2 fold change at N-Myc signature genes were statistically tested using the Mann–Whitney U test. Promoters with more than 2-fold increase relative levels of H3K27me3 were considered to be differentially expressed and were tested for enriched signatures using the hypergeometric test and the MSigDB database (Liberzon et al., 2011).
Genome_build: hg19/mm9

Submission date

Sep 07, 2016

Last update date

May 15, 2019

Contact name

Etienne Dardenne

E-mail(s)

etienne.dardenne@gmail.com

Organization name

Weill Cornell Medecine

Department

Pathology