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| Status |
Public on Dec 31, 2017 |
| Title |
dcl1dcl2dcl4_FACS_pollen |
| Sample type |
SRA |
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| Source name |
Pollen
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| Organism |
Arabidopsis thaliana |
| Characteristics |
ecotype: Col-0
tissue: Pollen
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| Growth protocol |
Plants were grown under long day conditions at 22 °C. Seeds were always surface sterilized with sodium hypochlorite, sowed on Murashige and Skoog (MS) medium and stratified for 3 days at 4ºC. Seedlings were transplanted to soil two weeks after germination and grown under long day conditions at 22 °C.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Pollen was collected in 1.5mL eppendorf tubes by vortexing open flowers in pollen extraction buffer (PEB, 10 mM CaCl2, 2 mM MES, 1 mM KCl, 1% H3BO3, 10%) for 3 min, followed by filtration through a 30um mesh (Partec/Sysmex) and centrifugation at 5,000g for 1 min. Purification of pollen expressing the GFP-miR845b sensor in dcl1-5/+ and dcl1/+,dcl2/dcl4 mutants was performed by vortexing open flowers in 1 mL of PEB and filtering through a 30um mesh before FACS. Pollen population was identified in SSC/FSC scatter plots, and GFP fluorescence was analyzed by excitation with a 488nm laser and detected with a 530/30 bandpass filter. Pollen total RNA was extracted using Trizol LS reagent (Life Technologies) by vortexing with glass beads for 5 minutes, and concentrated with Direct-zol columns (Zymo Research).
Small RNAs were purified by running total RNA on acrylamide gels (15% polyacrylamide, 7M urea) and performing size selection of approximately 18 to 30-nt region using a small RNA ladder (Zymo Research). The small RNA fraction was isolated from acrylamide gel plugs by grinding with a plastic pestle in Trizol LS (Life Technologies), and concentrated using Direct-zol columns (Zymo Research). Libraries were prepared using the TruSeq Small RNA Sample Preparation Kit (Illumina) following manufacturer instructions.
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| Library strategy |
ncRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Small RNA
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| Data processing |
After de-multiplexing, 36-nt reads were pre-processed by trimming the 3’ adapter and filtering collapsed reads according to length and quality.
Filtered reads were mapped to the Arabidopsis TAIR10 genome annotation (or GFP transgene) with bowtie reporting all multi-mappers.
Only perfect match reads were used for down-stream analysis, and reads mapped to multiple genomic locations where normalized by dividing non-redundant read counts by the number of genomic hits, and subsequently calculating the number of reads per million of filtered (18-30nt) and perfectly mapped reads.
Genome_build: TAIR10
Supplementary_files_format_and_content: txt files with 18-30nt reads and counts, and RPM of 20-24nt reads mapped to TAIR10 features
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| Submission date |
Oct 24, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Filipe Borges |
| E-mail(s) |
filipe.borges@inrae.fr
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| Organization name |
Institut Jean-Pierre Bourgin - INRAE
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| Street address |
Route de St-Cyr (RD10)
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| City |
Versailles |
| ZIP/Postal code |
78026 |
| Country |
France |
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| Platform ID |
GPL19580 |
| Series (2) |
| GSE106114 |
microRNA-triggered transposon small RNAs mediate genome dosage response (sRNA-Seq) |
| GSE106117 |
microRNA-triggered transposon small RNAs mediate genome dosage response |
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| Relations |
| BioSample |
SAMN07830375 |
| SRA |
SRX3318193 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2829915_dcl1dcl2dcl4_FACS_pollen.txt.gz |
1.4 Mb |
(ftp)(http) |
TXT |
| GSM2829915_dcl1dcl2dcl4_FACS_pollen_tair10.txt.gz |
135.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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