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| Status |
Public on Jul 21, 2021 |
| Title |
OA3 |
| Sample type |
SRA |
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| Source name |
OA
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| Organism |
Glycine max |
| Characteristics |
tissue: leaves
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| Treatment protocol |
Inoculation of soybean plants with BPMV vectors targeting Ssoah1 (pBPMV-OA) first occurred in Williams 82 soybean. Symptomatic plants were then used to rub inocualte Traff.
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| Growth protocol |
The modified system, used in this work and developed by Zhang (2010), uses bean pod mottle virus (BPMV) which is bipartite. The two viral segments consist of a 6 kb RNA1 segment (pBPMV-IA-R1M) encoding viral proteins needed for genome replication and a 3.6 kb RNA2 segment (pBPMV-IA-V1) that encodes viral proteins needed for capsid assembly and movement and contains the cloning site where the silencing construct was inserted. As described in the protocol by Whitham et al. (2016), the RNA1 and RNA2 segments were co-bombarded into 10-day old Williams 82 (PI 518671) seedlings using gold particles coated with plasmid DNA. Two inoculations were made per plant, with one on each unifoliate leaf. Prior to inoculations, unifoliate seedlings were forced to etiolate in the dark for 24 h. After inoculations, plants were sprayed with water and kept in plastic bags for 24 h to maintain humidity. The inoculated plants were placed in a growth chamber at 22 °C during the day and 20 °C at night on a 16 h photoperiod. Soil was checked daily to determine if water was needed, and the plants were fertilized once weekly with Miracle-GroÒ (Scotts Miracle-Gro Co.).
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| Extracted molecule |
total RNA |
| Extraction protocol |
A high concentration of total RNA was extracted from three leaves in three biological replicates from the pBPMV-OA and pBPMV-EV- containing plants. Leaves for RNA extractions were collected from the variety Traff, prior to inoculating soybeans symptomatic for BPMV with S. sclerotiorum in one experimental replicate of the expression experiment. The third trifoliate leaves were immediately frozen in liquid nitrogen. Two grams of leaf tissue per biological replicate was ground into fine powder using liquid nitrogen in a mortar and pestle and used in RNA extractions. A phenol chloroform extraction with a LiCl purification was performed as described in Aragão et al. (2013) to yield high RNA concentrations of 1.2-2.8 ug/ul.
A total amount of 2 μg total RNA per sample was used as input material for the small RNA library. Sequencing libraries were generated using NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (NEB, USA.) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, 3' SR Adaptor was directly and specifically ligated to 3' end of miRNA, siRNA and piRNA. After the 3' ligation reaction, the SR RT Primer hybridized to the excess of 3' SR Adaptor (that remained free after the 3' ligation reaction) and transformed the single-stranded DNA adaptor into a double-stranded DNA molecule. This step is important to prevent adaptor-dimer formation, besides, dsDNAs are not substrates for ligation mediated by T4 RNA Ligase 1 and therefore do not ligate to the 5´ SR Adaptor in the subsequent ligation step. 5´ends adapter was ligated to 5´ends of miRNAs, siRNA and piRNA. Then first strand cDNA was synthesized using M-MuLV Reverse Transcriptase (RNase H–). PCR amplification was performed using LongAmp Taq 2X Master Mix, SR Primer for Illumina and index (X) primer. PCR products were purified on an 8% polyacrylamide gel (100V, 80 min). DNA fragments corresponding to 140~160 bp (the length of small noncoding RNA plus the 3' and 5' adaptors) were recovered and dissolved in 8 μL elution buffer. At last, library quality was assessed on the Agilent Bioanalyzer 2100 system using DNA High Sensitivity Chips.
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| Library strategy |
ncRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Quality control：trimmomatic；
Mapping：bowtie
Further: Self-written script to organize the results.
Supplementary_files_format_and_content: .xls, mapping and normalization
For the empty files (not archived), are you referring to the processed data? The data files that are empty are the alignments to our target sequence from the RNA of plants containing an empty silencing vector. There were zero alignments to the target sequence in plants containing our empty silencing vector (all processed files containing "EV") because the plants did not contain the silencing construct.
Under the files that say "normalized" in their name please look in the RPM column. These are the raw reads; They were multiplied by the conversion factor to get the small RNA count. We are not showing differentially expressed genes of soybean here. Instead, we are looking for genes that correspond to a fungus, Sclerotinia sclerotiorum, that were expressed from an RNAi silencing construct.
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| Submission date |
Jul 14, 2021 |
| Last update date |
Jul 21, 2021 |
| Contact name |
Mehdi Kabbage |
| E-mail(s) |
[email protected]
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| Organization name |
University of Wisconsin Madison
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| Department |
Department of Plant Pathology
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| Lab |
583 Russell Labs 1630 Linden Dr.
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| Street address |
Department of Plant Pathology, 583 Russell Labs 1630 Linden Dr
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| City |
Madison |
| State/province |
Wisconsin |
| ZIP/Postal code |
53706 |
| Country |
USA |
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| Platform ID |
GPL28801 |
| Series (1) |
| GSE180131 |
Sequencing of siRNA from soybean containing silencing vectors and empty vectors |
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| Relations |
| BioSample |
SAMN20224497 |
| SRA |
SRX11449936 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5453777_OA3.MappedRreads.xls.gz |
14.9 Kb |
(ftp)(http) |
XLS |
| GSM5453777_OA3.antisense.xlsx |
75.1 Kb |
(ftp)(http) |
XLSX |
| GSM5453777_OA3.rpm_normalized.xls.gz |
39.5 Kb |
(ftp)(http) |
XLS |
| GSM5453777_OA3.sense.xlsx |
113.5 Kb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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