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Sample GSM1358031

Query DataSets for GSM1358031

Status

Public on Apr 01, 2015

Title

HCT-116 parental (A)

Sample type

SRA

Source name

Colon cancer cell line

Organism

Homo sapiens

Characteristics

cell line: HCT-116 parental cell line is sensitive for both OxPt and SN38

Treatment protocol

Oxaliplatin or SN-38 resistant cell lines were generated in our laboratory over a period of 8-10 months by continuous exposure to gradually increasing concentrations of drug. The cell lines were passaged three times at each drug concentration and cell vials were frozen at each increase in drug concentration. Prior to subsequent experiments, the cells were maintained in drug-free growth medium for at least 1 week.

Growth protocol

The cell lines HCT116 and HT29 were obtained from the NCI/Development Therapeutics Program, while LoVo was obtained from the American Tissue Culture Collection. Cells were maintained at 37 ˚C, 5% CO2 in RPMI 1640+Glutamax growth medium (Invitrogen, Naerum, Denmark) supplemented with 10% foetal calf serum (Invitrogen).

Extracted molecule

genomic DNA

Extraction protocol

Genomic DNA was isolated from the cell lines and clinical samples using DNeasy® Blood & Tissue Kit (QIAGEN) following manufacturer's protocol. The extracted genomic DNA was verified by agarose gel electrophoresis, and the concentration was estimated by NanoDrop (Thermo Scientific). Furthermore, BioAnalyzer 2100 DNA Chip was used to ensure the quality, quantity and integrity for the genomic DNA.
Five µg genome DNA from the cell line models and the clinical samples was digested by restriction enzyme, MspI (New England BioLabs) over night at 37 °C and QIAGEN Mini Purification kit was used to purify the digested products. End repair, adding A and adaptors, where the cytosines in the paired end adaptor sequence were methylated, was performed. The ligated product was subjected to size selection in 2% agarose gel (Bio-RAD) at 100 V for 2 hours. Agarose gel bands with the inserted genomic DNA size 40 ~ 110 bp and the inserted genomic DNA size 110 ~ 220 bp were excised, so that two libraries were generated from the MOMA3, MOMA4, MOMA5, MOMA7, MOMA8 and MOMA9 (one consisting of 40 ~ 110 bp target sequences and the other of 110 ~ 220 bp target sequences). The rest clinical samples and all cell line samples were generated with a single library with inserted DNA fragments with 40-300 bp. The DNA from the excised gel pieces was recovered by QIAGEN Gel Extraction Purification Kit, followed by bisulfite treatment using ZYMO EZ DNA Methylation-Gold kit. The resulting converted DNA was amplified by PCR and purified. The RRBS libraries were performed paired-end 50 nt sequencing with HiSeq 2000 (Illumina).

Library strategy

Bisulfite-Seq

Library source

genomic

Library selection

Reduced Representation

Instrument model

Illumina HiSeq 2000

Description

A
RRBS-seq

Data processing

Illumina Casava1.7 software used for basecalling.
The adaptor sequences were filtered out from the subsequent analysis and the resulting reads were aligned using Bismark software. Only uniquely mapped reads, which had restriction enzyme cutting site at the 5’ end were used to subsequent methylation analysis.
The sequencing depth and the percentage of methylated cytosines/total investigated cytosines for each C location were calculated. The genomic annotation information was based on the hg19 human genome (http://genome.ucsc.edu). Differentially methylated cytosines were identified by QDMR.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text file includes the significantly differented cytosines and the correpsonding methylation level (percentage) values for each sample in the colon cancer cell line models and the clinical samples.

Submission date

Mar 27, 2014

Last update date

May 15, 2019

Contact name

Jian Li

E-mail(s)

jianlij@gmail.com

Phone

45+40608651

Organization name

Aarhus University