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Sample GSM2829914 Query DataSets for GSM2829914
Status Public on Dec 31, 2017
Title dcl2dcl4_FACS_pollen
Sample type SRA
 
Source name Pollen
Organism Arabidopsis thaliana
Characteristics ecotype: Col-0
tissue: Pollen
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.
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.
 
Library strategy ncRNA-Seq
Library source transcriptomic
Library selection size fractionation
Instrument model Illumina NextSeq 500
 
Description Small RNA
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
 
Submission date Oct 24, 2017
Last update date May 15, 2019
Contact name Filipe Borges
E-mail(s) filipe.borges@inrae.fr
Organization name Institut Jean-Pierre Bourgin - INRAE
Street address Route de St-Cyr (RD10)
City Versailles
ZIP/Postal code 78026
Country France
 
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
Relations
BioSample SAMN07830376
SRA SRX3318192

Supplementary file Size Download File type/resource
GSM2829914_dcl2dcl4_FACS_pollen.txt.gz 1.5 Mb (ftp)(http) TXT
GSM2829914_dcl2dcl4_FACS_pollen_tair10.txt.gz 164.0 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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