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Sample GSM2472867

Query DataSets for GSM2472867

Status

Public on Jan 31, 2020

Title

pOsNAR2.1-OsNAR2.1 WGBS replicate 3

Sample type

SRA

Source name

DNA methylation of pOsNAR2.1-OsNAR2.1

Organism

Oryza sativa

Characteristics

tissue: shoot

Growth protocol

We grew plants water for 10 days after germination. The Nipponbare WT (NP), OsNAR2.1 RNAi and pUbi-OsNAR2.1 lines for MEDIP-seq were grown in plots at Nanjing Agricultural University in Nanjing, Jiangsu, 2014. Nanjing, Jiangsu is in a subtropical monsoon climate zone. Chemical properties of the soils in the plots included organic matter, 11.56 g/kg; total N content, 0.91 g/kg; available P content, 18.91 mg/kg; exchangeable K, 185.67 mg/kg; and pH 6.5. Basal applications of 30 kg P/ha (Ca(H2PO4)2) and 60 kg K/ha (KCl) were made to all plots 3 days before transplanting. N fertilizer as urea accounted for 40%, 30% and 40% of the total N fertilizer was applied prior to transplanting, at tillering, just before the heading stage, respectively.

Extracted molecule

genomic DNA

Extraction protocol

Genomic DNA were extracted from 10-day-old seedlings, and 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.
MeDIP sequecing in this study done by KangChen Bio-tech, Shanghai, China. Sequencing library preparation and data analysis was described as before (Ding et al 2015 ). Genomic DNA was sonicated to ~200 - 600bp with a Bioruptor sonicator (Diagenode). 800 ng of sonicated DNA was end-repaired, A-tailed, and ligated to single-end adapters following the standard Illumina genomic DNA protocol. After agarose size-selection to remove unligated adapters, the adaptor-ligated DNA was used for immunoprecipitation using a Rat monoclonal anti-5-methylcytosine antibody (Diagenode). For this, DNA was heat-denatured at 94°C for 10 min, rapidly cooled on ice, and immunoprecipitated with 1 μL primary antibody overnight at 4°C with rocking agitation in 400 μL immunoprecipitation buffer (0.5% BSA in PBS). To recover the immunoprecipitated DNA fragments, 100 μL of protein G magnetic beads (Life Technologies) were added and incubated for an additional 2 hours at 4°C with agitation. After immunoprecipitation, a total of five immunoprecipitation washes were performed with ice-cold immunoprecipitation buffer. A nonspecific Rat IgG immunoprecipitation was performed in parallel to methyl DNA immunoprecipitation as a negative control. Washed beads were resuspended in TE buffer with 0.25% SDS and 0.25 mg/mL proteinase K for 2 hours at 65°C and then allowed to cool down to room temperature. MeDIP and supernatant DNA were purified using Qiagen MinElute columns and eluted in 16 μL EB (Qiagen). 14 cycles of PCR were performed on 5 μL of the immunoprecipitated DNA using the single-end Illumina PCR primers. The resulting reactions were purified with Qiagen MinElute columns, after which a final size selection (300-700 bp) was performed by electrophoresis in 2% agarose. Libraries were quality controlled by Agilent 2100 Bioanalyzer. An aliquot of each library was diluted in EB to 5 ng/μL and 1 μL was used in real-time PCR reactions to confirm the enrichment for methylated region.

Library strategy

Bisulfite-Seq

Library source

genomic

Library selection

RANDOM

Instrument model

Illumina HiSeq 4000

Description

WTvspOsNAR2.1-OsNAR2.1_DMR.xls
pOsNAR2.1-OsNAR2.1 3

Data processing

Whole-genome bisulfite sequencing
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.
MeDIP sequecing
After the sequencing platform generated the sequencing images, the stages of image analysis and base calling were performed using Off-Line Basecaller software (OLB V1.8). After passing Solexa CHASTITY quality filter, the clean reads were aligned to rice genome (Ensembl Plants Oryza sativa Japonica, IRGSP-1.0) using BOWTIE software (V2.1.0, B Langmead, et al. 2012). MeDIP peaks were identified by MACS2(Zhang et al 2008), and statistically significant MeDIP-enriched regions (peaks) were identified by comparison to a Poisson background model (Cut-off q-value=10-2). Differentially Methylated Regions (DMRs) in MeDIP dataset was identified by diffReps(Shen et al 2013) (p < 0.0001), and promoter-associated DMRs were annotated with IRGSP-1.0 database.
Genome_build: IRGSP1
Supplementary_files_format_and_content: Processed data were stored in excel files, which included differentially methylated regions from WGBS and enriched peaks from MeDIP.

Submission date

Jan 31, 2017

Last update date

Jan 31, 2020

Contact name

Xiaorong Fan

E-mail(s)

xiaorongfan@njau.edu.cn

Organization name

Nanjing Agricultural University

Street address

Tongwei Road 1