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Sample GSM4982814

Query DataSets for GSM4982814

Status

Public on Nov 26, 2021

Title

bovine_FGO_DNAme_rep1

Sample type

SRA

Source name

oocyte

Organism

Bos taurus

Characteristics

developmental stage: full_grown oocyte

Extracted molecule

genomic DNA

Extraction protocol

STEM-Seq library preparation The whole genome bisulfite libraries for bovine and porcine FGO were constructed with STEM-seq as previously described (Zhang et al., 2018a). Briefly, the oocytes were firstly lysed with 10μL lysis buffer and 1μL protease K at 55°C for 3 hours. The protease K was inactivated at 72°C for 40 minutes. After lysis, the spike-in λ-DNA (Promega; D150A) was added with a mass ratio of 1:200. Then the mixture was treated and then purified with the bisulfite conversion reagent with the EpiTect Fast Bisulfite Conversion Kit (Qiagen; 59824) following the manufacture’s instruction, with a modified protocol: denature for 8 min at 95 °C, incubate at 60 °C for 25 min, and repeat the procedure once. Finally, the purified converted DNA was subject to TELP library construction process as previously reported (Peng et al., 2015).

Library strategy

Bisulfite-Seq

Library source

genomic

Library selection

RANDOM

Instrument model

HiSeq X Ten

Description

Cow_FGO_DNAme_merge.rmdup.bismark.cov.gz

Data processing

Basecalls performed using CASAVA version 1.8
All STEM-seq datasets were firstly treated with cutadapt v1.11 (Martin, 2011) and the low-quality reads and adaptors were removed before mapping. Then all filtered reads were aligned to different genomes (rat, rn6; cow, bosTau8; pig, susScr11) using Bismark (v0.7.0；bowtie2 2.1.0) (Krueger and Andrews, 2011) with default parameters. Multi-mapped reads and PCR duplicates were removed also with the Bismark. The unique mapped reads were used for CpG methylation calling with bismark_methylation_extractor. For every single CpG site, only the CpG sites that were covered at least 3 times were used for further analysis. The spiked-in Lamda DNA (1:200) was used for conversation rate calculation.
All RNA-seq datasets were mapped to the different genomes (rat: rn6; cow: bosTau8; pig: susScr11). All genomes were downloaded from UCSC (citation) by Tophat v2.1.1 (Trapnell et al., 2009). Gene expression was then calculated according to refFlat database by cufflinks 2.2.1 (Trapnell et al., 2012).
All reads were firstly processed by TrimGalore (v.0.6.4) with default parameters to remove the poor qualitied reads and also the adapters. Then the filtered reads were mapped to different genomes (rat: Rn6; cow: bosTau8; pig: susScr11) by Bowtie2 (v2.2.5) (Langmead and Salzberg, 2012) with the default parameters. All multiple mapped reads as well as the duplicates were removed with MarkDuplicates.jar. Then all unique mapped reads were used for the downstream RPKM (reads per kilobase per million of sequenced reads) calculation. The Pearson correlation was generated for each sample with two biological replicates with 10 kb bins. The replicates with good reproducibility were pooled for downstream analysis.
Supplementary_files_format_and_content: The cov txt files contains the chr, start positon, methylated CG count, unmethylated CG count. The txt files for FPKM contain gene names and the FPKM value for all stages.

Submission date

Dec 21, 2020

Last update date

Nov 26, 2021

Contact name

Wei Xie

E-mail(s)

xiewei121@tsinghua.edu.cn

Organization name

Tsinghua University

Street address

Zhongguancun north street