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Sample GSM1334471 Query DataSets for GSM1334471
Status Public on Jun 01, 2018
Title RRBS_LCC0
Sample type SRA
 
Source name Breast cancer cell line derived from MCF-7
Organism Homo sapiens
Characteristics tissue: Breast cancer
cell line: LCC0
Treatment protocol The LCC0 is derived from MCF-7 presenting estrogen-dependent and tamoxifen-responsive phenotype. LCC1 was established from the estrogen-dependent parent cell line MCF-7 through prolonged withdrawal from potent estrogenic stimuli both in vivo and in vitro. The above in vivo selected cell line was further passaged in ovary-comprised nude mice and re-established in vitro to generate a new cell line, MCF-7/LCC1, which is estrogen-independent, but tamoxifen-responsive as its parent cell line. Furthermore, a new cell hormone-independent, tamoxifen-resistant subline, LCC2 was developed through growth of LCC1 in incrementally increased dosages of tamoxifen in vitro. And a new cell hormone-independent, cross-resistance to tamoxifen and fluvestrant subline, LCC9 was developed through growth of LCC1 in incrementally increased dosage of fluvestrant.
Growth protocol The LCC cell lines were cultured in DMEM/F12 without phenol red, supplemented with 5% DCC and 1% penicillin/streptomycin. The cells were maintained at 37°C in a humidified atmosphere of 95% ambient air and 5% CO2.
Extracted molecule genomic DNA
Extraction protocol Genomic DNA was isolated from the LCC cell lines 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. Subsequently, the genomic DNA was subject to digestion of RNase A (Invitrogen), and then purified by ethanol precipitation for subsequent analyses.
For the RRBS analysis, 5 µg genome DNA from the LCC0, LCC1, LCC2 and LCC9 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 DNA size 40 ~ 110 bp and the inserted DNA size 110 ~ 220 bp were excised, so that two libraries were generated from each sample (one consisting of 40 ~ 110 bp target sequences and the other of 110 ~ 220 bp target sequences). DNA from the two 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, following purification, the RRBS libraries were generated by performing 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 estrogen-dependent and tamoxifen sensitive
LB-NB9
processed data file: LCCs_RRBS_processed_data.txt
Data processing Base calling software: Illumina Casava1.7
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 LCC cell line model.
 
Submission date Feb 25, 2014
Last update date May 15, 2019
Contact name Jian Li
E-mail(s) jianlij@gmail.com
Phone 45+40608651
Organization name Aarhus University
Department Institute of Biomedicine
Lab 10
Street address The Bartholin Building
City Aarhus
ZIP/Postal code DK-8000
Country Denmark
 
Platform ID GPL11154
Series (2)
GSE55343 Integrative Analysis of Tamoxifen-resistant Cell Line Models Based on Sequencing Genomes, Transcriptomes and Epigenomes [seq]
GSE55383 Integrative Analysis of Tamoxifen-resistant Cell Line Models Based on Sequencing Genomes, Transcriptomes and Epigenomes
Relations
BioSample SAMN02664984
SRA SRX476014

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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