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| Status |
Public on Jun 11, 2020 |
| Title |
BT-549_RRBS |
| Sample type |
SRA |
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| Source name |
BT-549 Cell line
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| Organism |
Homo sapiens |
| Characteristics |
cell line: BT-549
cell type: breast cancer cell line
method: RRBS
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| Growth protocol |
The following cell lines: MDA-MB-231, MDA-MB-157, MDA-MB-361 , MDA-MB-453, and MDA-MB-468, were cultured in DMEM media supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, and non-essential amino acids. MDA-MB-134 cells were cultured in DMEM, 20% FBS, 1 mM sodium pyruvate, and non-essential amino acids. MDA-MB-436 were cultured in DMEM, 10% FBS, 10 μg/mL insulin, and 16 μg/mL glutathione. 2LMP and DY36T2 cells were cultured in Improved MEM and 10% FBS. HCC70, HCC1187, HCC1937, HCC1143, HCC1569, HCC1599, HCC1954, HCC38, ZR-75-1, and ZR-75-30 cells were cultured in RPMI media, supplemented with 10% fetal bovine serum, 10 mM HEPES, 1 mM sodium pyruvate, and 4.5 g/l glucose. BT-20 cells were cultured in EMEM, 10% FBS, 1 mM sodium pyruvate, and non-essential amino acids. BT-474, BT-549 and T-47D cells were cultured in RPMI, 10% fetal bovine serum, 10 mM HEPES, 1mM sodium pyruvate, 4.5g/l glucose, and 10µg/ml insulin. MCF-7 cells were cultured in EMEM, 10% fetal bovine serum, 1mM sodium pyruvate, non-essential amino acids and 10µg/ml insulin. SUM159 and SUM149 cells were cultured in Ham’s F12, 5% fetal bovine serum, 1µg/ml hydrocortisone, and 5µg/ml insulin. SUM102 cells were cultured in Ham’s F12, 5 μg/ml insulin, 1 μg/ml hydrocortisone, 10 ng/ml EGF, 5 mM ethanolamine, 10 mM HEPES, 5 μg/ml transferrin, 10 nM triiodothyronine (T3), 50 mM sodium selenite, 1 g/l BSA (2% FBS added for first 24 h after plating to allow cells to attach). SKBR-3 cells were cultured in McCoy’s media and 15% FBS. T-47D cells were cultured in Cells were passaged and harvested at 80% confluence.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted with the Qiagen DNeasy Blood and Tissue Kit.
Libraries were constructed using the protocol described in Varley KE, Gertz J, Bowling KM, et al. Dynamic DNA methylation across diverse human cell lines and tissues. Genome Res. 2013;23(3):555‐567. doi:10.1101/gr.147942.112 (PMCID: PMC3589544).
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
We aligned these reads to a modified reference genome sequence that was created to reflect both the reduced representation of the genome due to the MspI restriction digest as well as the sodium bisulfite conversion which creates a T in the sequencing reads rather than a C at all unmethylated bases. To create this reference, we first parsed the hg19 reference genome to identify all of the MspI restriction fragments <500 bp. We then isolated the 36-bp ends of these fragments into a fasta file and converted every C in the reference to a T and recorded the position of these reference cytosines in the name of the reference sequence. To achieve optimal alignment that is not biased by the methylation state of a molecule, we also created a copy of our sequencing read files, converted every C in the read to a T, and recorded the position of these read cytosines in the name of the read. We then used bowtie (Langmead et al. 2009) to align these converted reads to the custom reference sequence and required that the alignment be optimal and unique in the reference and only align to the proper strand (bowtie options –best, -m 1, norc).
We then parsed the alignment file and the encoded read and reference names to determine how many reads covered each reference cytosine position.
For each position with at least 10 reads of coverage, we calculated the percentage of those reads that contained a C and reported the percent of reads that were methylated in the processed data file.
Genome_build: hg19
Supplementary_files_format_and_content: Matrix of percent methylated values
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| Submission date |
Jun 10, 2020 |
| Last update date |
Jun 11, 2020 |
| Contact name |
Katherine E Varley |
| E-mail(s) |
kt.varley@hci.utah.edu
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| Organization name |
Huntsman Cancer Institute University of Utah
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| Department |
Department of Oncological Sciences
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| Lab |
Varley Lab
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| Street address |
2000 Circle of Hope, Room 3719
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| City |
Salt Lake City |
| State/province |
UT |
| ZIP/Postal code |
84112 |
| Country |
USA |
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| Platform ID |
GPL10999 |
| Series (2) |
| GSE152202 |
Reduced representation bisulfite sequencing (RRBS) of breast cancer cell lines |
| GSE152205 |
Integrated analysis of DNA methylation, chromatin accessibility, transcription factor binding, and gene expression in large collections of breast cancer cell lines and patient tumors |
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| Relations |
| BioSample |
SAMN15198532 |
| SRA |
SRX8520767 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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