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| Status |
Public on Sep 24, 2020 |
| Title |
embryo_sRNA_rep3 |
| Sample type |
SRA |
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| Source name |
embryo_sRNA
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| Organism |
Oryza sativa |
| Characteristics |
genotype: Nipponbare
Stage: embryo
molecule subtype: 15–30-nt sRNAs
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| Growth protocol |
Rice seeds of Nipponbare (japonica), Zhongxian 3037 (indica), Huanghuazhan (indica) as well as rdr6-2 and mel1-4 were sterilized with 5% NaClO containing 0.05% Tween-20 for 45 min. The seeds were then germinated and grown in either a paddy field in Changping District, Beijing, China (116.42°, 40.10°) from May to October or in a greenhouse with 40% humidity and daily cycles of 14?h of light at 32?°C and 10?h of dark at 26?°C. Tillers at a proper stage were harvested for collection of spikelets and male germ cells.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Libraries for sRNA-seq were constructed as previously described (Yang et al, 2016) with minor modifications. Briefly, 3'adaptor, DNA oligonucleotide with 5'adenylation, was obtained using 5′ DNA Adenylation Kit (NEB, E2601L). Total RNA was ligated to 3 adaptor using T4 RNA ligase 2, truncated KQ (NEB, M0373L). Following annealing with reverse transcription primer, RNA was ligated to 5'RNA adaptor using T4 RNA ligase 1 (NEB, M0204L). After reverse transcription and PCR amplification, 135–160-bp products were gel-purified. Libraries were pair-end sequenced on an Illumina HiSeqX-Ten platform.
Total RNA was extracted using TRIzol (Takara, 9109). MgCl2 (final concentration: 0.2 M) was added to prevent loss of sRNAs with low GC content. The extracted RNA was stored at -80°Cin LoBind tubes (Eppendorf, 022431021).
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| Library strategy |
ncRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
HiSeq X Ten |
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| Data processing |
sRNA-seq:Low quality reads were removed and 3' adapter sequences were trimmed using cutadapt2.7. Clean sRNA reads in sizes of 18–30 nt were mapped to the rice reference genome (MSU v7) using bowtie, allowing no mismatches. 21-nt and 24-nt PHAS loci were identified and characterized by PhaseTank and Unitas as previously described using all 21-nt and 24-nt genome-matched reads from spikelets and male germ cells. The overlapped results between these two programs are considered as our final annotated phasiRNA loci.For annotation of phasiRNA loci that were not previously identified, only those loci with higher phase score than lowest score of previous PHAS catalog(phase score=10) were considered as the newly identified loci.For examining the dependence of phasiRNA accumulation on MEL1 or OsRDR6, the read counts of each phasiRNA from three biological replicates of wild-type and mutant plants were compared using edegR. PhasiRNAs with a ≥ 2-fold reduction in the mutant and an FDR ≤ 0.05 were considered to be dependent on OsRDR6 or MEL1.
RNA-seq:Low quality reads were removed and adapters were trimmed using Trimmomatic (v0.36). Clean RNA reads were then mapped to the reference genome (MSU v7) with default parameters using TopHat v2.1.1. The expression level of each gene was normalized to Fragments Per Kilobase of transcript per Million mapped reads (FPKM). Differential gene expression analysis was performed using Cuffdiff. Genes with a ≥ 1.5-fold change and an FDR ≤ 0.05 were identified as differentially expressed genes.
Degradome：All of annotated phasiRNAs were aligned to rice cDNA sequences downloaded from rice genome database (MSU v7) using psRobot_tar(-ts 3.5 –fp 2 –tp 17). Genes (include TEs) that pair with phasiRNAs with mispair scores ≤ 3.5 were selected as an initial pool of phasiRNA targets.Mispaired score was calculated by psRobot as the following rules: 1) Mismatches, gaps or bulges are evaluated with a penalty of +1 2); G:U pairs are evaluated a penalty of +0.5; 3) Half penalty scores will be evaluated outside the defined essential sequence region(2-17).Cleavage products of predicted phasiRNA targets were then searched in degradome libraries using psRobot_deg. We characterized our predicted phasiRNA targets to five categories(Category 0-Category 4) base on degradome tags distribution of 10th and 11thcleavage sites as previous described38 Only predicted phasiRNA target filled with category 0 and category 1 were considered phasiRNA targets in this study for further analysis. When a gene has ≥ 4 degradome tags between the 10th and 11th nucleotides from the 5'ends of the aligned phasiRNAs and these degradome tags accounted for ≥ 10% of the total degradome tags along this gene locus, the gene was defined as a target of the aligned phasiRNA.
Genome_build: MSU v7
Supplementary_files_format_and_content: For RNA-Seq processed dataset，our provided file is the expression matrix of genes . The expression level of lncRNAs are estimated as FPKM. For sRNA-seq /degradome processed datasets, our provided files contain genome match small RNA sequences and their counts.
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| Submission date |
May 04, 2020 |
| Last update date |
Sep 24, 2020 |
| Contact name |
Yijun Qi |
| E-mail(s) |
qiyijun@mail.tsinghua.edu.cn
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| Organization name |
Tsinghua University
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| Department |
School of Life Sciences
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| Street address |
NO.1 Qinghuayuan
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| City |
Beiing |
| ZIP/Postal code |
100084 |
| Country |
China |
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| Platform ID |
GPL24468 |
| Series (1) |
| GSE149800 |
21-nt phasiRNAs direct target mRNA cleavage in rice male germ cells |
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| Relations |
| BioSample |
SAMN14827324 |
| SRA |
SRX8244875 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4512860_Embryo_sRNA_Rep3.uni.txt.gz |
3.2 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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