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Sample GSM3926474

Query DataSets for GSM3926474

Status

Public on Jul 01, 2021

Title

WT_leaf_smallRNA-seq

Sample type

SRA

Source name

WT leaf

Organism

Oryza sativa Japonica Group

Characteristics

variety: Oryza sativa ssp. japonica; variety ZhongHua11
tissue: leaf
developmental stage: 50days

Growth protocol

The Epi-lf mutant was identified in a screen of an anther-cultured population of Japonica rice (Oryza sativa ssp. japonica; variety ZhongHua11; ZH11) in 2005. The mutant phenotype of Epi-lf was stably inherited over more than 20 generations. The mutant and WT rice plants were planted in paddy fields under natural conditions or in glasshouses with a day temperature of 30°C and a night temperature of 24°C.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted from different tissues using TRIzol reagent (Thermo Fisher Scientific). The first-strand cDNA was synthesized using 1 μg RNA and ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo), according to the manufacturer’s instructions, using 29 PCR cycles to amplify the LF transcripts and 23 PCR cycles for Actin1. The RT-PCR analysis was repeated three times with similar results. Genomic DNA was isolated from the flag leaves of WT and tillers with different phenotypes of Epi-lf using the CTAB method. Briefly, 1 μg of genomic DNA was treated with sodium bisulphite using the Qiagen EpiTect Bisulfite kit, according to the manufacturer’s instructions. The treated DNA was dissolved in 15 mL distilled water, and 3 μL of this solution was used as the template in a 50-μL PCR (94°C for 4 min; followed by 40 cycles of 94°C for 30 s , 55°C for 30 s and 60°C for 30 s; with a final 60°C for 10 min).The PCR products were purified and cloned into pEASY-T5-Blunt Zero Cloning Kit vectors.
RNA libraries were prepared for sequencing using standard Illumina protocols

Library strategy

ncRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina HiSeq 2500

Description

All_FPKM.csv

Data processing

For Bisulfite-Seq data, the adaptor sequences and low-quality reads were trimmed using trim_galore(https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). The clean data were mapped against genome using bismark(v0.19.0). CGmaptools(v0.1.1) was used to calculate the methylation level of the 2-kb sequences upstream and downstream of all genes and transposable elements (http://rice.plantbiology.msu.edu/). A personal Python script was used to visualize the methylation level.
For RNA-seq data, the adaptor sequences and low-quality reads were trimmed using trim_galore(https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/).The clean reads were mapped against replaced genome using Tophat2 (v2.1.1)[5] with default parameters. Cufflinks (v2.0.0) was used to quantify the relative expression of the genes and calculate any differentially expressed genes (Fold change ≥ 1, adjusted p-value<0.01). To visualize the RNA-seq expression level, bedtools was used to calculate the coverage and normalize the data per million mapped reads. A Python script was used to visualize the data.
For smallRNA-seq data, The adaptor sequences and low-quality RNA-seq reads were trimmed using trim_galore, and reads shorter than 18 nt or longer than 30 nt were discarded. MicroRNA (http://www.mirbase.org/ftp.shtml), transfer RNA (http://rice.plantbiology.msu.edu/analyses_search_tRNA.shtml), ribosomal RNA (Rice Genome Annotation MSU7.0), small nucleolar RNA, and small nuclear RNA (https://www.arb-silva.de/no_cache/download/archive/release_132/Exports/) sequences were also removed. The remaining clean reads were aligned to the replaced MSU7.0 rice genome using Bowtie (v 1.1.1), allowing 2 mismatches and requiring mapping to a unique site. The siRNA enrichment was calculated in 10-kb windows with normalized reads, and only windows with at least three reads were further analysed. To visualize the level of siRNA expression, bedtools was used to calculate the coverage and normalize the data per million mapping reads. A Python script was used to visualize the data.
Genome_build: About 7-kb sequence containing LOC_Os01g41370 was amplified from ZH11 and sequenced. This sequence was used to replace the LOC_Os01g41370 sequence in the MSU7.0 rice genome assembly, which was identified using BLASTn. The replaced MSU7.0 rice genome was used in our analysis.

Submission date

Jul 02, 2019

Last update date

Jul 01, 2021

Contact name

mao fei

E-mail(s)

maofei@fafu.edu.cn

Organization name

Fujian Agriculture and Forestry University

Department

National Engineering Research Center of JUNCAO