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| Status |
Public on Mar 24, 2022 |
| Title |
RRBS DNA methylation data of bovine embryonic fibroblast cells |
| Organism |
Bos taurus |
| Experiment type |
Methylation profiling by high throughput sequencing
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| Summary |
We investigated the effects of one carbon metabolites supplementation on early embryonic development. To this end, the Bovine Embryonic Tracheal Fibroblast cell lines (EBTr; NBL-4; ATCC CCL-44) were cultured under different levels of glucose and OCM (folic acid, choline chloride, vitamin B12, and L-methionine).
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| Overall design |
The EBTr cells were cultured in Eagle’s Minimum Essential Medium, supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin and 0.11 g/L Na pyruvate (Sigma). Treatments were arranged as a completely randomized design with a two glucose levels [1 g/L (low) or 4.5 g/L (high)] × 3 OCM levels [control, 2.5X, and 5X]. Control medium contained basal concentrations of folate (0.001g/L), choline (0.001 g/L), vitamin B12 (4 µg/L), and methionine (0.015 g/L). One-carbon metabolites (folic acid, choline chloride, vitamin B12, and L-methionine) were supplemented to the media to achieve 2.5 or 5 times.
For all treatments, cells were plated into T-75 culture flasks and allowed to become 90% confluent. Once target confluence was reached, cells were removed from the flask and cell pellets (1 × 10^6) were frozen in cryo vials (n = 3 tubes/trt) and shipped to the University of Minnesota Genomics Center. The DNA was extracted from the cell pellets with the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) and quantified using the PicoGreen DNA Quantification Kit (Invitrogen). Libraries (n = 18) were prepared from extracted DNA (20 ng/µL) using the NuGEN Ovation RRBS Methyl-Seq Kit (Tecan Genomics, Redwood City, CA). Libraries were bisulfite converted and PCR amplified prior to sequencing with NovaSeq S Prime in the 150 paired-end reads mode (Illumina, San Diego, CA).
After sequencing, raw data quality control was performed using the FastQC v0.11.8 and MultiQC v1.9 software. Adapter trimming and low-quality bases (Phred score < 20) were filtered out using Cutadapt v.2.10. NuGEN’s diversity trimming was carried out to remove diversity adaptors. Trimmed reads were mapped to the bovine reference genome (UCSC - bosTau8, Illumina IGenome) using the Nextflow methylseq pipeline v1.5. Differentially methylated cytosines (DMC) were identified by pair-wise comparison using edgeR v. 3.24.3, and considered significant for each of the contrasts when the P-value cutoff < 0.01.
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| Contributor(s) |
Diniz WJ, Crouse MS, Caton JS, Claycombe-Larson KJ, Lindholm-Perry AK, Reynolds LP, Dahlen CR, Borowicz PP, Ward AK |
| Citation(s) |
35281844, 35392625 |
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| Submission date |
Jul 19, 2021 |
| Last update date |
Apr 19, 2022 |
| Contact name |
Wellison J. S. Diniz |
| Organization name |
Auburn University
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| Department |
Animal Sciences
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| Lab |
Livestock Genomics & Bioinformatics
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| Street address |
CASIC Building 559 Devall Drive Auburn University, 205
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| City |
Auburn |
| State/province |
Alabama |
| ZIP/Postal code |
36849 |
| Country |
USA |
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| Platforms (1) |
| GPL26012 |
Illumina NovaSeq 6000 (Bos taurus) |
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| Samples (18)
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| Relations |
| BioProject |
PRJNA747992 |
| SRA |
SRP328920 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE180362_Normalized_Cytosinelevel.csv.gz |
2.6 Mb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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