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| Status |
Public on Mar 09, 2017 |
| Title |
LXD_L-1 |
| Sample type |
SRA |
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| Source name |
rice anther
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| Organism |
Oryza sativa Japonica Group |
| Characteristics |
material: diploid rice
floret length/mm: 2.6-2.8
pollen/embryo sac stage: pre-meiotic interphase (PMA)
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| Treatment protocol |
Autotetraploid rice, 02428-4x, and its diploid rice, 02428-2x, were used in this study. 02428-4x was developed from the chromosome doubling of 02428-2x by colchicine and was self-crossed for more than 25 generations in our lab. They were planted at the experimental farm of South China Agricultural University (SCAU) under field conditions. Anthers with four pollen development stages, including pre-meiotic interphase (PMA), meiosis (MA), single microspore stage (SCP) and bi-cellular pollen stage (BCP), were collected from 02428-4x and 02428-2x based on the floret length and DAPI fluorescence (Additional file 2: Table S23). The ovary tissues were collected with their corresponding embryo sac development stages (i.e. megasporocyte formation stage (MF), megasporocyte meiosis stage (MM), functional megaspore formation stage (FMF) and eight-nucleate embryo sac developing-stage (EES)). All the samples were stored at 4 °C for cytological observation and -80 °C for RNA isolation.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from the anthers using Trizol reagent (Invitrogen, CA, USA) following the manufacturer’s procedure. The total RNA quantity and purity were analysis by Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA) with RIN number >7.0. Approximately 1 μg of total RNA were used to prepare small RNA library according to the protocol of TruSeq small RNA sample preparation Kits (Illumina, San Diego, USA). Then we performed the single-end sequencing (36 bp) on an Illumina Hiseq2500 at the LC-BIO (Hangzhou, China) according to the manufacturer’s protocol. After the Illumina sequencing, the raw reads were subjected to the Illumina pipeline filter (Solexa 0.3), and then the dataset was further processed with an in-house program, ACGT101-miR (LC Sciences, Houston, Texas, USA) to detected the known and novel miRNA.
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| Library strategy |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
PMA-2x
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| Data processing |
The raw reads were subjected to the Illumina pipeline filter (Solexa 0.3), and then the dataset was further processed with an in-house program, ACGT101-miR (LC Sciences, Houston, Texas, USA) to remove adapter dimers, junk, low complexity, common RNA families (rRNA, tRNA, snRNA, snoRNA) and repeats.
Subsequently, unique sequences with length in 18-25 nt were mapped to rice precursors in miRBase 20.0 (ftp://mirbase.org/pub/mirbase/CURRENT/) by BLAST search to identify known miRNAs
The unmapped sequences were BLASTed against the rice genomes, and the hairpin RNA structures containing sequences were predicated from the flank 120 nt sequences using RNAfold software (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi).
We used PhaseTank to systemically characterize the 21 and 24nt phasiRNAs (Guo et al. 2015). Then the confidence phasiRNAs with phasing scores higher than or equal to 25 were selected (Zhai et al. 2015), and filtered the phasiRNAs that were triggered by miR2118 and miR2275 families. Finally, it yielded 1117 loci generating 21nt-phasiRNAs and 190 loci generating 24nt-phasiRNAs. PhasiRNAs with P-value (Fisher exact test) < 0.05 and log2 (fold change ratio) >1 were considered as differentially expressed phasiRNAs.
To find out the siRNAs associated with the transposable elements, we followed the method described by Zhang et al. (2015). Firstly, we filtered the reads that did not match miRNA, rRNA, tRNA, snRNA, or snoRNA, and then selected the reads which mapped to the transposable elements of rice reference genome. These subsequent of siRNAs were considered as TEs-siRNAs, and used for further analysis. The classifications of TEs-siRNAs were done by Li et al. (2016). The differentially expressed TEs-siRNAs were identified by the conditions of P-value (Fisher exact test) < 0.05 and log2 (fold change ratio) >1.
Supplementary_files_format_and_content: Tab-delimited text files include RPM values for each Sample
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| Submission date |
Nov 23, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
andyslee Li |
| E-mail(s) |
andyslee@126.com
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| Organization name |
South China Agricultural University
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| Lab |
State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources
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| Street address |
Wushan 483
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| City |
Guangzhou |
| ZIP/Postal code |
510642 |
| Country |
China |
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| Platform ID |
GPL18525 |
| Series (1) |
| GSE90479 |
Analysis of small RNAs revealed differential expressions during the development of pollen and embryo sac and their targets may associate with fertility in autotetraploid rice |
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| Relations |
| BioSample |
SAMN06051315 |
| SRA |
SRX2371441 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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