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Sample GSM4982950 Query DataSets for GSM4982950
Status Public on Nov 26, 2021
Title rat_MII_H3K4me3_CR_rep1
Sample type SRA
 
Source name oocyte
Organism Rattus norvegicus
Characteristics developmental stage: In vivo matured mataphase II oocyte
antibody: H3K4me3
Extracted molecule genomic DNA
Extraction protocol CUT&RUN library preparation and sequencing CUT&RUN was performed as previously reported (Skene et al., 2018; Skene and Henikoff, 2017) with modifications. Briefly, after removing the zona pellucida with Tyrode’s solution (Sigma-Aldrich, T1788) or 0.5% (m/v) Protease from Streptomyces griseus (Sigma-Aldrich, P8811), mammalian oocytes and early embryos were incubated with Concanavalin-coated magnetic beads (Polyscience, 86057) for 10 min at room temperature on a thermomixer at 400 rpm. Samples were then incubated with primary antibody at a ratio of 1:100 at 4℃ overnight on a thermomixer at 400 rpm. The next day, after washing for one time, beads were incubated with pA-MNase (to a final concentration of 400-700ng/mL) (a gift from Steven Henikoff lab) at 4℃ for 3 hours on a thermomixer at 400 rpm. After two times of washing, targeted digestion was performed by adding 2μL of 100mM CaCl2 for 30 mins on ice, followed by termination by adding an equal volume of 2 × stop buffer. Samples were then incubated at 37℃ for 20 mins for fragment releasing. The total samples or supernatants were digested with Proteinase K (NEB, P8107S) and purified using phenol:chloroform:isoamyl alcohol (25:24:1, v/v) followed by ethanol purification at -80℃ overnight. The next day, DNA was purified and subjected to Truseq library preparation using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645S). Sequencing was done using the HiSeq X Ten system (Illumina) according to the manufacturer’s protocol.
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model HiSeq X Ten
 
Description rat_MII_H3K4me3_CR.bw
Data processing Library strategy: Cut&Run
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
City Beijing
ZIP/Postal code 100084
Country China
 
Platform ID GPL24688
Series (1)
GSE163620 The conservation and divergence of epigenitic reprogramming during mammalian early development
Relations
BioSample SAMN17128706
SRA SRX9710892

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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