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| Status |
Public on Aug 01, 2014 |
| Title |
NOK cl1 AkataBX-1 cl1 p28 EBV- |
| Sample type |
SRA |
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| Source name |
NOK EBV-
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| Organism |
Homo sapiens |
| Characteristics |
tissue: gingiva
cell type: hTert immortalized normal oral keratinocytes derived from gingiva
infection state: Transiently-infected EBV-
passage: p28
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| Treatment protocol |
Uninfected parental cells, vector control (pTVRF=PTURF), EBV-infected, and EBV-negative transiently-infected clones were seeded at 1x10^6 cells in a T75 flask. DNA was harvested 3 days after seeding in using phenol/chloroform extraction, followed by ethanol precipitaiton of the DNA.
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| Growth protocol |
Human telomerase (hTERT)-immortalized normal oral keratinocytes (NOK, kindly gifted by Dr. Karl Munger) were grown in keratinocyte serum free media (KSFM) supplemented with human epidermal growth factor and bovine pituitary extract (Life Technologies) in a humidified CO2 incubator at 37oC. Cells were passaged once a week using trypsinization at a 1:4 split.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted from cells by DNA lysis buffer (100mM NaCl, 10mM Tris pH 8, 25mM EDTA, 0.5% SDS, 0.1mg/ml proteinase K). DNA extracts were incubated overnight at 56oC. DNA was purified by 2 rounds of phenol/chloroform and precipitated with 2 volumes of ethanol and 1/10 volume of 3M sodium acetate solution.
Libraries were prepared by the Zymo Epiquest service. RRBS libraries were prepared from 200-500 ng genomic DNA digested with 60 units of TaqI and 30 units of MspI (NEB) sequentially. Size-selected TaqI-MspI fragments (40–120 bp and 120–350 bp) were filled in and 3’-terminal-A extended, extracted with Zymo Research DNA Clean & Concentrator™ kit. Ligation to pre-annealed adapters containing 5’- methyl-cytosine instead of cytosine (Illumina) was performed using the Illumina DNA preparation kit and protocol. Purified, adaptor ligated fragments were bisulphite-treated using the EZ DNA Methylation-Direct™ Kit (Zymo Research). Preparative-scale PCR was performed and DNA Clean & Concentrator™ -purified PCR products were subjected to a final size selection on a 4% NuSieve 3:1 agarose gel. SYBR-green-stained gel slices containing adaptor-ligated fragments of 130–210 bp or 210–460 bp in size were excised. Library material was recovered from the gel (Zymoclean™ Gel DNA Recovery Kit) and sequenced on the Illumina HiSeq platform.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Sequence reads from bisulphite-treated EpiQuest libraries were identified using standard Illumina base-calling (bcl2fastq version 1.8.3).
Sequence reads from bisulphite-treated EpiQuest libraries were identified using standard Illumina base-calling (bcl2fastq version 1.8.3). Bismark version 0.7 was used for alignment and data was analyzed using a proprietary (Zymo) pipeline implemented in Python (version 2.7) for methylation calling and further downstream analysis. Residual cytosines (Cs) in each read were first converted to thymines (Ts), with each such conversion noted for subsequent analysis. A reference sequence database was constructed from the 50-bp ends of each computationally predicted MspI-TaqI fragment in the 40–350 bp size range. All Cs in each fragment end were then converted to Ts (only the C-poor strands are sequenced in the RRBS process; the converted reads were aligned to the converted reference by Bowtie. The number of mismatches in the induced alignment was then counted between the unconverted read and reference, ignoring cases in which a T in the unconverted read is matched to a C in the unconverted reference. For a given read, the best alignment was kept if the second-best alignment had 2 more mismatches, otherwise the read was discarded as non-unique. The methylation level of each sampled cytosine was estimated as the number of reads reporting a C, divided by the total number of reads reporting a C or T. Fisher’s exact test or t-test were performed for each CpG site which has at least 5 reads covered. CpG were classified as hypermethylation or hypomethylated (meth ratio 0.01 – 0.33) based on methylation difference in the uninfected sample compared to all other samples. Thus, a hypomethylated CpG refers to its state in the uninfected cell line. Strongly hyper and hypomethylated CpGs had a meth ratio between 0.33 – 1.00). Also, promoter, gene body and CpG island annotations were added to each CpG.
Data were filtered using the following specifications: * pvalue <= 0.05
Second filtering of data based on * Site position is not part of the vector sample (ie, vector and uninfected had same methylation call) * classification is the same across ebv+ and all ebv- samples. (e.g., if a record is ebv+ stronglyHypermeth, then all ebv- values must also be stronglyHypermeth)
For the EBV+ sample, reads that did not align to EBV were aligned to the Akata EBV strain (KC207813).
Genome_build: hg19
Supplementary_files_format_and_content: 6 Bed files are provided for visualization of CpG methylation calls (human) in uninfected, vector, EBVpositive, and three EBV-negative clones (cl1 ,cl3, cl4). 5 CSV files are included with CpG methylation data filtered by p<= 0.05 for vector, EBVpos, and three EBV-negative clones (cl1, cl3, cl4) as CSV files. This data is compared to the uninfected sample. 1 CSV files with final processed data p<= 0.05 with methylation differences being unique for EBV pos and three EBV negative, transiently infected clones compared to uninfected and vector controls, EBV_CpG_methylation data (CSV file).
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| Submission date |
Jul 28, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Rona S. Scott |
| E-mail(s) |
rscott1@lsuhsc.edu
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| Phone |
318-675-6263
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| Organization name |
Louisiana State University Health Sciences Center-Shreveport
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| Department |
Microbiology and Immunology
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| Street address |
1501 Kings Highway
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| City |
Shreveport |
| State/province |
LA |
| ZIP/Postal code |
71130 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE59842 |
Genome wide DNA methylation of Epstein-Barr virus infected immortalized normal oral keratinocytes |
| GSE59843 |
Genome wide DNA methylation and expression profiling of Epstein-Barr virus infected immortalized normal oral keratinocytes |
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| Relations |
| BioSample |
SAMN02943223 |
| SRA |
SRX664897 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1447377_ebv_neg_cl1.bed.gz |
54.2 Mb |
(ftp)(http) |
BED |
| GSM1447377_ebv_neg_cl1.csv.gz |
3.7 Mb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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