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Sample GSM3091848

Query DataSets for GSM3091848

Status

Public on Apr 08, 2019

Title

CaCO2_Avi_Smad4_BiotinChIP

Sample type

SRA

Source name

CaCO2 cells

Organism

Homo sapiens

Characteristics

tissue: colon epithelial cell line (colorectal adenocarcinoma)
genotype: CaCO2_Avi_Smad4_Biotin cells
cell line: CaCO-2

Growth protocol

Caco-2 cells were cultured in DMEM (Gibco 11995-065), containing 10% FBS (Gibco 26140-095) and 1% penicillin and streptomycin (Invitrogen 15140-122). Cells stably expressing both avi-tagged Smad4 and BirA were selected with media containing 2 ug/ml puromycin and 0.4 mg/ml Hygromycin B, respectively. Cells were fed 24 hours prior to harvest at ~95% confluency.

Extracted molecule

genomic DNA

Extraction protocol

To perform SMAD4 ChIP in Caco-2 cells, each 100 μl pellet were incubated with 1 ml of cross linking solution containing equal amount of DSG (Thermo 20593), DSS (Thermo 21655) and EGS (Thermo 21565) with a final concentration of 2mM NHS ester (reactive groups). Cells were rocked for 20 min at room temperature, after which fresh formaldehyde was added to a concentration of 1.22% formaldehyde, and rocked for an additional 20 min. The cross-linking reaction was stopped with 125 mM glycine, washed with PBS and, nuclei extracted using Downs’ homogenizer and Farnham lysis buffer (5 mM PIPES, pH 8.0, 85 mM KCl, 0.5% NP40, and protease inhibitor cocktails). IP material was subsequently handled as described above for conventional ChIP of sonicated genomic DNA (a final concentration of 0.2% SDS in the sonicates). The diluted sonicates were then incubated with pre-blocked (0.5% BSA/PBS) Streptavidin beads (Invitrogen) overnight at 4°C. The beads were serially washed in low salt (0.1% SDS, 0.1% Sodium Deoxycholate, 1% Triton X-100, 150 mM NaCl, 1mM EDTA, and 20 mM HEPES, pH 8.0), high-salt (0.1% SDS, 0.1% Sodium Deoxycholate, 1% Triton X-100, 500 mM NaCl, 1mM EDTA, and 20 mM HEPES, pH 8.0), LiCl biuffer (250 mM LiCl, 0.5% Sodium Deoxycholate, 0.5% NP40, 1mM EDTA, and 20 mM HEPES, pH 8.0), and final wash buffer (1mM EDTA, and 20 mM HEPES, pH 8.0), and incubated at 65 °C for 6h in reverse cross linking buffer (0.1mM NaHCO3, 0.01% SDS).
Rubicon Genomics ThruPLEX DNA-seq Kit (Illumina)

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

NextSeq 550

Description

CaCO2_Avi_Smad4_BiotinChIP.bw

Data processing

FastQC (version 0.11.3) was used to check the quality of raw sequencing reads (fastq)
Alignment: RNA-seq: TopHat2, version 2.1.0; ATAC-seq and ChIP-seq: Bowtie2, version 2.2.6
RNA-seq: Gene and transcript level computation done with CuffQuant, version 2.2.1
RNA-seq: Normalized abundance measurements generated by CuffNorm, version 2.2.1, calculate FPKM values using quartile normalization
RNA-seq: CuffDiff, version 2.2.1, geometric normalization, per-condition dispersion
ATAC-seq and ChIP-seq: Deeptools bamCoverage (v2.4.2, duplicate reads ignored, RPKM normalized and extended reads) was used to generate bigwig files from bam files.
ATAC-seq and ChIP-seq: Model-based Analysis of ChIP-Seq (MACS 1.4.1) was used for peak calling and to generate bed files from aligned reads. Shiftsize parameter was used for MACS based on the fragment size of Pippin Prep.
Genome_build: mm9/hg19

Submission date

Apr 10, 2018

Last update date

Aug 23, 2019

Contact name

Michael P Verzi

Organization name

Rutgers, the State University of New Jersey

Department

Genetics

Lab

Verzi