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Sample GSM4982942

Query DataSets for GSM4982942

Status

Public on Nov 26, 2021

Title

rat_FGO_H3K36me2_STAR_rep2

Sample type

SRA

Source name

oocyte

Organism

Rattus norvegicus

Characteristics

developmental stage: full_grown oocyte
antibody: H3K36me2

Extracted molecule

genomic DNA

Extraction protocol

STAR ChIP-seq library preparation and sequencing STAR ChIP-seq was performed as previously described (Zhang et al., 2016). Briefly, samples were lysed followed by fragmented by micrococcal nuclease (MNase, Sigma, N3755-200UN) at 37 °C for 5 min. After being terminated, the supernatant containing chromatin was incubated with the primary antibody at 4 °C overnight with rotation. The next day, 100 μg of Protein A/G dynabeads (mixed at 1:1, Thermo Fisher Scientific) was added to each sample and incubated at 4 °C for 2–3 h with rotation. The beads were then washed five times with 150 μl RIPA buffer and once with 150 μl LiCl buffer. For each sample, beads were resuspended with 28 μl H2O, 1 μl 10× Ex-Taq buffer (Takara, RR006B) and 1 μl proteinase K (NEB, P8107S) and incubated at 55 °C for 90 min, followed by incubation at 72 °C for 40 min to inactivate the proteinase K. Samples were then subjected to Truseq library preparation using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645S). STAR ChIP-seq libraries were sequenced using the HiSeq X Ten system (Illumina) according to the manufacturer’s protocol.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

HiSeq X Ten

Description

rat_FGO_H3K36me2_STAR.bw

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