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Sample GSM4512903 Query DataSets for GSM4512903
Status Public on Sep 24, 2020
Title prophase_I_degradome_rep3_run1
Sample type SRA
Source name prophase_I_degradome
Organism Oryza sativa
Characteristics genotype: Nipponbare
Stage: meiocyte (prophase I)
molecule subtype: poly (A)+ RNAs
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.
Extracted molecule total RNA
Extraction protocol Libraries for mRNA-seq were constructed using the Smart2-seq method40. Briefly, total RNA was used for reverse transcription and cDNA second strand synthesis using SuperScript II reverse transcriptase (Invitrogen,18064-14) and template-switching oligos. After PCR preamplification and purification with AMPure XP beads (Beckman, A63880), the products were subjected to fragmentation and adaptor ligation using TruePrepTM DNA Library Prep Kit (Vazyme, TD503). PCR amplification was then performed with index-containing primers and 300–800-bp products were purified with AMPure XP beads. Libraries were pair-end sequenced on an Illumina HiSeqX-Ten platform by Annoroad Gene Technology (Beijing).
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).
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model HiSeq X Ten
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.
Submission date May 04, 2020
Last update date Sep 24, 2020
Contact name Yijun Qi
Organization name Tsinghua University
Department School of Life Sciences
Street address NO.1 Qinghuayuan
City Beiing
ZIP/Postal code 100084
Country China
Platform ID GPL24468
Series (1)
GSE149800 21-nt phasiRNAs direct target mRNA cleavage in rice male germ cells
BioSample SAMN14827308
SRA SRX8244918

Supplementary file Size Download File type/resource
GSM4512903_leptotene_deg_rep3_run1.uni.txt.gz 11.9 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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