 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Apr 22, 2022 |
| Title |
WGBS_Control_#2 |
| Sample type |
SRA |
| |
|
| Source name |
Day 105 fetal skin fibroblast primary cell
|
| Organism |
Bos indicus x Bos taurus |
| Characteristics |
conception method: Artificial insemination
developmental stage: D105 fetus
disease status: Healthy
cell type: fibroblast
|
| Treatment protocol |
For bovine fetuses, artificial insemination was used to generate the control group and assisted reproductive technologies (ART) were used to generate the ART group following the protocol previous described (PMID: 23751783). The large offspring syndrome (LOS) group was defined as individuals from the ART group with body weight greater than 97th centile of controls.
|
| Growth protocol |
Day 105 Bos taurus indicus (B. t. indicus; Brahman breed) x Bos taurus taurus (B. t. taurus; Angus breed) F1 hybrid fetuses were generated by our laboratory following the protocol previous described (PMID: 23751783) and used as tissue donors. Fetal skins were collected to establish fibroblast primary cell line using protocol adapted from a publication (PMID: 24198123). Fibroblast cells were cultured in bovine embryonic fibroblast medium (89% (v/v) DMEM (Gibco, 11885084), 10% (v/v) fetal bovine serum (Atlanta Biologicalsa, S11150H), and 1% (v/v) Antibiotic-antimycotic (Gibco, 15240062)) at 38.5°C with 5% CO2.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fibroblasts were lysed in lysis buffer (0.05 M Tris-HCl (pH 8.0), 0.1 M EDTA, and 0.5% (w/v) SDS) with proteinase K (Fisher BioReagents, BP1700) at 55°C for four hours. Genomic DNA was extracted with Phenol:Chloroform:Isoamyl Alcohol (SIGMA, P3803) following the manufacturer’s instructions. The concentration of DNA was measured by using a NanoDrop® ND-1000 Spectrophotometer (Thermo Fisher Scientific) and DNA integrity was confirmed by electrophoresis on a 0.7% agarose gel. Genomic DNA samples were stored at -20°C.
This WGBS was conducted by CD Genomics (Shirley, NY, USA). Information on library preparation and sequencing obtained from the company is as follows: For WGBS library preparation, 1 ug of genomic DNA was fragmented by sonication to a mean size of approximately 200-400 bp. Fragmented DNA was end-repaired, 5'-phosphorylated, 3'-dA-tailed and then ligated to methylated adapters. The methylated adapter-ligated DNAs were purified using 0.8× Agencourt AMPure XP magnetic beads and subjected to bisulfite conversion by ZYMO EZ DNA Methylation-Gold Kit (zymo). The converted DNAs were then amplified using 25 μl KAPA HiFi HotStart Uracil+ ReadyMix (2X) and 8-bp index primers with a final concentration of 1 μM each. The constructed WGBS libraries were then analyzed by Agilent 2100 Bioanalyzer and quantified by a Qubit fluorometer with Quant-iT dsDNA HS Assay Kit (Invitrogen), and finally sequenced on Illumina Hiseq X ten sequencer. 0.1-1% lambda DNA were added during the library preparation to monitor bisulfite conversion rate.
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
HiSeq X Ten |
| |
|
| Data processing |
Duplicated reads generated during PCR and sequencing were removed from raw sequencing reads using the clumpify function of BBMap (version 38.90). The remaining raw reads were trimmed for adapter sequences and low quality bases using trimmomatic (version 0.39) with parameters ‘ILLUMINACLIP:adapter_seq:2:30:10:1:true LEADING:20 TRAILING:20 AVGQUAL:20 MAXINFO:0:0.5’. Trimmed reads were aligned to the bovine genome using bismark (version 0.23.0) with parameters ‘-X 900 --unmapped --ambiguous --non_bs_mm’. Trimmed reads were also aligned to lambda phage genome to determine bisulfite conversion rates. Samtools (version 1.13) was used to convert, sort, filter, and index bam files. MarkDuplicates function of picard (version 2.25.5) was used to further remove duplicated reads after alignment. Read groups were added for each samples using AddOrReplaceReadGroups function of picard. Variants identified in bull semen genomic sequencing data and variants in bovine acquired from the 1000 bull genome project, namely ARS1.2PlusY_BQSR_v3.vcf.gz, served as known variants to identify genomic variants in WGBS data. Indel realignment was performed using RealignerTargetCreator and IndelRealigner functions of BisSNP (version 1.0.1). Base quality recalibration was carried out using BisulfiteCountCovariates and BisulfiteTableRecalibration functions of BisSNP (version 0.82.2) since these functions are missing in version 1.0.0 and 1.0.1. Parameters used for BisulfiteCountCovariates were ‘-cov ReadGroupCovariate -cov QualityScoreCovariate -cov CycleCovariate -baqGOP 30’. Genomic variants were identified using BisSNP (version 1.0.1) with default setting expect that ‘-bsRate’ was changed to bisulfite conversion rate observed from lambda phage genome alignment for each sample. BisSNP identified variants were filtered by its VCFpostprocess function with parameter ‘-windSizeForSNPfilter 0’. Additionally, genomic variants were identified using BS-SNPer (version 1.0) with parameters ‘-minhetfreq 0.1 --minhomfreq 0.85 --minquali 15 --mincover 5 --maxcover 1000 --minread2 2 --errorate 0.02 --mapvalue 20’. M-bias plots were generated using bismark and the first 3 bases of R1 reads and the first 4 bases of R2 reads showed biased CpG methylation level, thus these bases were excluded from downstream analyses. CpG methylation information were extracted from the bam files using bismark_methylation_extractor function of bismark with parameters ‘-p --ignore 3 --ignore_r2 4 --comprehensive --no_header –gzip --bedGraph --buffer_size 50% --cytosine_report’. Statistical analyses were conducted using R package hummingbird (version 1.2.0) with parameter ‘minCpGs = 10, minLength = 100, maxGap = 300’ to identify differentially methylated regions (DMRs) between LOS and Control groups. DMRs with at least 15% difference in methylation level (both gain and loss of methylation) and at least 2 mean read coverage at CpG sites were reported. The sex chromosomes were not analyzed to circumvent confounding created by X chromosome inactivation associated DNA methylation.
Genome_build: ARS-UCD1.2
Supplementary_files_format_and_content: Read coverage and DNA methylation percentage for CpG sites in BEDGRAPH format.
|
| |
|
| Submission date |
Feb 21, 2022 |
| Last update date |
Apr 22, 2022 |
| Contact name |
Rocío Melissa Rivera |
| E-mail(s) |
riverarm@missouri.edu
|
| Organization name |
University of Missouri, Columbia
|
| Department |
Division of Animal Sciences
|
| Street address |
920 East Campus Drive
|
| City |
Columbia |
| State/province |
MO |
| ZIP/Postal code |
65211 |
| Country |
USA |
| |
|
| Platform ID |
GPL31972 |
| Series (2) |
| GSE197129 |
Allele-specific aberration of imprinted domain chromosome architecture associates with large offspring syndrome [WGBS] |
| GSE197130 |
Allele-specific aberration of imprinted domain chromosome architecture associates with large offspring syndrome |
|
| Relations |
| BioSample |
SAMN26137946 |
| SRA |
SRX14242066 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5909680_WGBS_Control__2_CpG_coverage.bedgraph.gz |
160.5 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM5909680_WGBS_Control__2_CpG_methylation.bedgraph.gz |
171.4 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
