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Sample GSM4512907 Query DataSets for GSM4512907
Status Public on Sep 24, 2020
Title microspore_degradome_rep3
Sample type SRA
 
Source name microspore_degradome
Organism Oryza sativa
Characteristics genotype: Nipponbare
Stage: microspore
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 degradome sequencing were constructed based on the PARE method with considerable modifications that allow use of low-input RNAs. In detail, 5’ RNA adaptor was first heated at 75 °C for 3 min and then immediately chilled on ice for 2 min. For 5’ RNA adapter ligation, total RNA (3 μL) was added to a solution composed of 1 μL of T4 RNA ligase buffer, 1 μL of 10 mM ATP, 0.5 μL of RNasin, 0.5 μL of 10 μM treated RNA adaptor, 3 μL of PEG8000 (50% v/v), and 1 μL of T4 RNA ligase 1 and incubated at room temperature for 1 h. Then 1 μL of 10 mM dNTP and 0.5 μL of 10 μM reverse transcription primer Oligo(dT)VN were added. After incubating at 75°C for 5 min and chilling on ice for 2 min, 5 μL of first strand RT buffer, 5 μL of 5 M betaine (Sigma, 61962), 1.25 μL of 0.1 M DTT, 0.9 μL of 1/6 M MgCl2, 0.5 μL of Ribonuclease inhibitor (Promega, N2511), and 1 μL of SuperScriptIII reverse transcriptase (Invitrogen, 18080-085) were added for reverse transcription. The condition for reverse transcription was 44°C for 1 h followed by 15 cycle of 50°C for 2 min and 44°C for 2 min. After reverse transcription, the products were purified with 2 × AMPure XP beads (v/v). Then the products (20 μL) were pre-amplified using GXL DNA polymerase (Takara, R050A) with 5’ biotin-labelled forward primer F1 and reverse primer R1 in a solution composed of 10 μL of 5 × GXL buffer, 4 μL of 2.5 mM dNTP, 1.5 μL of 10 μM F-primer, 1.5 μL of 10 μM R-primer, 2 μL of GXL DNA polymerase, 11 μL of ddH2O. The PCR was performed at 98°C for 2 min followed by 20 cycle of 98°C for 10 s, 60°C for 15 s, 68°C for 3 min and then 68°C for 5 min. After PCR, 1 μL of Exonuclease I (NEB, M0293L) and 5.7 μL of 10 × Exonuclease I buffer were added to the sample and the mixture was incubated at 37°C for 1 h. Then 19 μL of 4 × binding buffer (40 mM Tris-HCl, pH 8.0, 2 mM EDTA, 4 M NaCl) was added and the mixture was incubated with DynabeadsMyOne Streptavidin C1 beads (Invitrogen, 65001) at room temperature for 30 min with rotation. The beads were washed once with 1 × TWB buffer (10 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween-20 v/v) and 3 times with 1 × EBT buffer (10 mM Tris-HCl, pH 8.0, 0.02% Triton X-100 v/v) and then digested with MmeI (NEB, R0637L) in a solution composed of 3 μL of 10 × NEB buffer 2, 3 μL of 0.5 mM SAM, 2 μL of Mme I and 22 μL of ddH2O at 37°C for 2 h with rotation. The beads were washed once with 1 × TWB buffer and 3 times with 1 × EBT buffer. The DNA products bound to the beads were ligated to 3’ DNA adaptor in a solution composed of 3 μL of 2 × quick ligase reaction buffer, 1 μL of 10 mM adaptor, 1 μL of quick ligase (NEB, M2200L) and 25 μL of ddH2O at room temperature for 30 min with rotation. The 3’ DNA adapter was prepared by annealing of two DNA oligos in 1 × annealing buffer (10 mM Tris-HCl pH 7.5, 0.1 mM EDTA, 50 mM NaCl). The beads were washed once with 1 × TWB buffer and 3 times with 1 × EBT buffer and resuspended in 30 μL of 0.05% Triton X-100. The samples were incubated at 80°C for 30 min with rotation to elute the DNA products. Then the DNA products were subjected to final PCR amplification using GXL DNA polymerase and primers F2 and R2. PCR was performed at 98°C for 2 min followed by 22 cycle of 98°C for 10 s, 60°C for 15 s, 68°C for 20 s and then 68°Cfor 5 min. The final products were resolved on a PAGE gel and ~140-bp bands were excised and purified for sequencing 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).
 
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
E-mail(s) qiyijun@mail.tsinghua.edu.cn
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
Relations
BioSample SAMN14827329
SRA SRX8244922

Supplementary file Size Download File type/resource
GSM4512907_NP_microspore_degradome_rep3.uni.txt.gz 1.4 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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