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| Status |
Public on May 03, 2022 |
| Title |
WGBS_US_SLOS_#6_Tongue |
| Sample type |
SRA |
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| Source name |
Tongue
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| Organism |
Bos taurus |
| Characteristics |
tissue: Calf tongue
breed: Charolais
Sex: Male
country: United States (US)
conception method: Artificial insemination/natural mating
disease status: SLOS
tissue: Tongue
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| Treatment protocol |
For Spain animals, the control group (ES_Control) were conceived by artificial insemination using frozen-thawed semen from one bull among synchronized cows on the day of presumptive estrus. In vitro produced animals were generated using slaughterhouse oocytes and semen from the same bull as controls. Following fertilization, embryos were separated in two different groups: one culture group (ES_ART) composed of synthetic oviductal fluid (SOF) media supplemented with bovine serum albumin during the 7-8 days of culture and another group (ES_RF) composed of SOF media supplemented with bovine oviductal fluid (NaturARTs-BOF-EL, Embryocloud, Spain) for the first 4 days and bovine uterine fluid (NaturARTs-BUF-ML, Embryocloud) for the following days. Embryos (blastocysts and expanded blastocysts) were vitrified on day 7 or 8 of culture and stored until use. Recipients were synchronized and on day 6 to 8 after presumptive estrus, each cow received one thawed embryo.
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| Growth protocol |
US_Control animals were purchased from the University of Missouri Foremost Dairy Research Center and used as tissue donors. Tissue samples of stillborn US_SLOS animals were collected by their owners, veterinarians, or our collaborators and shipped to us. Animals from Spain were generated as described previously (PMID: 32781545) and used as tissue donors.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Blood cells and tissues 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 (for blood cells) or overnight (for tissues). 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.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
HiSeq X Ten |
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| 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. The dataset of known variants in bovine, namely ARS1.2PlusY_BQSR_v3.vcf.gz, was acquired from the 1000 bull genome project and served as reference 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) in various comparisons. DMRs with at least 15% difference in methylation level (both gain and loss of methylation) and at least 4 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.
Assembly: ARS-UCD1.2
Supplementary files format and content: Read coverage and DNA methylation percentage for CpG sites in BEDGRAPH format.
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| Submission date |
Mar 21, 2022 |
| Last update date |
May 04, 2022 |
| Contact name |
Rocío Melissa Rivera |
| E-mail(s) |
riverarm@missouri.edu
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| Organization name |
University of Missouri, Columbia
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| Department |
Division of Animal Sciences
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| Street address |
920 East Campus Drive
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| City |
Columbia |
| State/province |
MO |
| ZIP/Postal code |
65211 |
| Country |
USA |
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| Platform ID |
GPL24230 |
| Series (1) |
| GSE199084 |
Spontaneous and ART-induced large offspring syndrome: similarities and differences in DNA methylome |
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| Relations |
| BioSample |
SAMN26851834 |
| SRA |
SRX14533613 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5963856_WGBS_US_SLOS__6_Tongue_CpG_coverage.bedgraph.gz |
137.7 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM5963856_WGBS_US_SLOS__6_Tongue_CpG_methylation.bedgraph.gz |
145.8 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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