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Sample GSM1617365 Query DataSets for GSM1617365
Status Public on Dec 20, 2016
Title Carcinoma_3 - H3K4me2_ChIPseq
Sample type SRA
Source name Primary colorectal carcinoma, snap-frozen tissue
Organism Homo sapiens
Characteristics tissue: Primary colorectal carcinoma
chip antibody: H3K4me2 (Millipore, catalog# 07-030, lot# 2019745)
patient: 3
Growth protocol Sections of snap-frozen tissues were either micrococcal nuclease treated or cross-linked with 1% formaldedyde for 20 minutes and sonicated for ChIPseq experiments followed b ChIP with 10ug of antibody. HCT116 cells were cross-linked with 1% formaldehyde for 20 min at 37oC, followed by ChIP with 10 µg CNOT3 for ChIPseq. For RNAseq experiments cells were washed with PBS and total RNA was extracted with TRIzol reagent (Invitrogen) and the RNeasy Mini Kit (Qiagen). RNA was treated with the Turbo DNA-free kit (Ambion) to remove genomic.
Extracted molecule genomic DNA
Extraction protocol ChIPseq: Lysates were clarified from micrococcal nuclease digested or sonicated or and histone-DNA or CNOT3-DNA complexed were isolated with antibody.
RNAseq: Total RNA was extracted with TRIzol reagent (Invitrogen) and the RNeasy Mini Kit (Qiagen). RNA was treated with the Turbo DNA-free kit (Ambion) to remove genomic DNA.
For ChIPseq:ThruPLEX-FD kits (Rubicon Genomics) was used and 50 bp single-end reads were sequenced on a HiSeq2000 instrument (Illumina).
For RNAseq: TruSeq RNA Sample Preparation Kit (Illumina) was used according to the manufacturer's instructions and 75 bp single-end reads were sequenced on an Illumina NextSeq 500 instrument.
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 2000
Description Carcinoma_3_from_patient_3
10 sections of 10 μm thickness were scraped into plastic tubes and treated with 0.2 U micrococcal nuclease (Sigma Aldrich) for 8 min at 37oC, followed by ChIP with 10 µg H3K4me2 Ab. DNA libraries were prepared using ThruPLEX-FD kits (Rubicon Genomics) and sequenced on a HiSeq2000 instrument (Illumina)
Data processing Alignment: For ChIPseq experiments we used Bowtie2 (2.1.0) with settings (--no-unal). For RNAseq we used Tophat, version 2.0.9, default options.
ChIPseq analyses: For peak calling we used MACS2 using Q:0.01.
RNAseq analyses: normalized and differential expression: Cufflinks, version 2.2.1; settings: -b, -u, and default; Cuffmerge was used with reference gtf and default settings; Cuffquant was used with default settings; Cuffnorm was used with default settings; Cuffdiff was used with -b, -u and default settings.
Genome_build: Hg19
Supplementary_files_format_and_content: ChIPseq: Wiggle files were generated by NPS software using parameters by default. Bed files (the 5 columns are 1: chromosome, 2: start location, 3: end location, 4: peak number, 5: score).
Supplementary_files_format_and_content: RNAseq: Individual samples have an FPKM gene tracking file from Cufflinks, estimating transcript abundance for that sample, along with confidence intervals. There are two processed data matrices that apply to all the samples. There exists a differential expression table, prepared using Cuffdiff with an FDR of 5%, which contains average normalized FPKM values for Controls infected with a non specific NS_shRNA construct (Scr) and CNOT3-depleted by infection with shRNA_#3 construct (silenced), as well as fold change in expression, P-values, and Q-values corrected for multiple hypothesis testing.
Submission date Feb 23, 2015
Last update date May 15, 2019
Contact name Paloma Cejas
Phone 6178406690
Organization name Dana Farber Cancer Institute
Department Medical Oncology
Street address 450 Brookline Ave
City Boston
State/province MA
ZIP/Postal code 02215
Country USA
Platform ID GPL11154
Series (1)
GSE66216 Nucleosome dynamics in human colorectal cancer specimens reveal activation of a CNOT3-regulated pathway of embryonic stem cell self-renewal
BioSample SAMN03366677
SRA SRX886381

Supplementary file Size Download File type/resource
GSM1617365_3_CARCINOMA.H3K4me2.peaks.bed.gz 102.7 Kb (ftp)(http) BED
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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