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Sample GSM4885513

Query DataSets for GSM4885513

Status

Public on Sep 15, 2021

Title

HN_WYG7-HN WGBS

Sample type

SRA

Source name

DNA methylation of HN_WYG7-HN

Organism

Oryza sativa

Characteristics

tissue: shoot
genotype: wild type

Treatment protocol

Plants were plants in the field fertilized at a rate of 300 kg N/ha as HN field, no N fertilizer as NN field and the field without N fertilizer (NN) since 2009.

Growth protocol

For plants grow in the field, all materials were grown in plots in the Agricultural Experiment Station of Zhejiang University, Changxing, Zhejiang. Changxing is located in a subtropical monsoon climate zone. The pH of the soil is 7.2. Potassium, phosphate was fertilization at usual levels in the fields (225kg/ha and 450kg/ha). Plots size was 2 × 2.5 m and the seedlings planted in a 10 × 10 array.

Extracted molecule

genomic DNA

Extraction protocol

For rice grow in the field, all materials were grown in plots in the Agricultural Experiment Station of Zhejiang University, Changxing, Zhejiang. Samples collected in the mature.
Samples shipped to the Anoroad Genome company(Beijing) for whole-genome bisulfite sequencing(WGBS). WGBS librarie preparation, sequencing and data analysis was described as before (??). WGBS libraries are prepared for subsequent cluster generation starting from sample DNA through adaptor ligation, library purification, and quantification. Input gDNA (5 μg) is fragmented by sonication. The fragments are blunt-ended and phosphorylated, and a single ʹAʹ nucleotide is added to the 3ʹ ends of the fragments in preparation for ligation to a methylated adapter that has a single-base ʹTʹ overhang. The ligation products are purified and size-selected by agarose gel electrophoresis. Size-selected DNA is bisulfite-treated and purified. The treated DNA is PCR-amplified to enrich the fragment that have adapters on both ends. The final purified product is then quantitated prior to cluster generation. The WGBS libraries will be sequenced using Illumina HiSeq2500 /4000 Analyzer according to the manufacturer’s instructions.

Library strategy

Bisulfite-Seq

Library source

genomic

Library selection

RANDOM

Instrument model

Illumina HiSeq 4000

Description

HN0NWT_HNHNWT_gene_intersect_dmr.dataset
HN0NWT_HNHNWT_dmr_zone.bed

Data processing

After downloading raw data from sequencing machine, in order to avoid alignment error, the raw reads will be filtered and trimmed to obtain clean reads. First, remove polluted adapter reads (remove the reads with the number of contaminants in the base more than 5bp) . Second, remove the reads with base calling quality Qemo), and third, remove reads with the percentage of N >5% (For pair-end sequencing, if one end is removed, both ends will be removed). After filtering, the available data were compared with the reference genome Oryza_sativa.IRGSP-1.0.32 to obtain the alignment results using Bismark (v0.9.0) (K Felix, et al. 2011). The uniquely mapped reads will be used to call the methylated cytosines(C) in highly enriched regions.Valid coverage of methylated cytosine, i.e. the percentage of methylated C among all C on genome, is associated with methylation level. C sites with read depths less than 5 are eliminated firstly. Then for each C site, valid coverage is calculated by for all C or C within each pattern (CpG, CGH, and CHH). Coverage is the count of C sites with more than 5 depth divided by total number of C site by its pattern. For each C site, the methylation level (%) is calculated by: 100* (reads supportted methylation)/ (total reads depth of this site). For methylated region, methylation level (%) is calculated by: 100*methylation level of all C sites in this region/Total number of C sites in this region. The conversion ratio of Lambda DNA is around 99.5% to 99.6% in all samples.
DMR for (1) The read coverage of each C locus > 4; (2) The total number of C loci in each DMR > = 5; (3) The length of DMR > = 100bp;(4) The difference of methylation level between groups was greater than or equal to 0.3;(5) The CPG site with p-value less than 1e-10 was reserved as the final DMR.
Based on the results of DMR genome annotation, the number of genes with overlapping promoter (1KB upstream) and gene body region
Genome_build: IRGSP1
Supplementary_files_format_and_content: Processed data were stored in txt files, which included differentially methylated region in dmr_zone file, and gene name in gene_intersect_dmr file.

Submission date

Nov 05, 2020

Last update date

Sep 15, 2021

Contact name

Xiaorong Fan

E-mail(s)

xiaorongfan@njau.edu.cn

Organization name

Nanjing Agricultural University

Street address

Tongwei Road 1