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Sample GSM5368489

Query DataSets for GSM5368489

Status

Public on Jun 01, 2024

Title

prors1 (LUC)_2

Sample type

SRA

Source name

26-d-old rbp-ko-1, grown under LD conditions

Organism

Arabidopsis thaliana

Characteristics

genotype: prors1 (LUC)

Growth protocol

The Arabidopsis plants were grown on soil for 25 days under long-day conditions (16 h light, 8 h dark)

Extracted molecule

total RNA

Extraction protocol

Total plant RNA was extracted from 25-d-old plants grown under LD condition using the Trizol reagent (Invitrogen, Carlsbad, USA) and dissolved in RNase-free H2O. To make siRNA library, 30 μg RNA was mixed with equal volume of 2× RPA loading buffer (10 ml of 98% formamide, 200 µl 2 mM EDTA, 10 mg bromophenol blue, 10 mg xylene cyanol) and denatured at 94℃ for 3 min. RNA samples were separated on 15% TBE-PAGE by running at 120 v for 2 h at 4℃, the gel was then stained with 5 µl of Syber Gold (Invitrogen, S11494) for 5 min in 50 ml of 1× TBE buffer (89 mM Tris base, 89 mM boric acid, 1 mM EDTA, pH 8.0). The gel band corresponding to 17-30 nt sRNA was cut and ground in a low-bind 1.5-ml tube with a RNase-free pestle in 500 μl of 0.3 M NaCl. Then the tube was incubated at -80℃ for 30 min at 4℃ overnight with slow rotation. The gel slurry was filtered through a Spin-X filter (Corning, Cat. 8162) by centrifugation at 20,000 ×g for 1 min at 4℃. The flow through was transferred to two 1.5-ml tubes, each contain 250 μl flow through and was added with 625 µl ethanol, 25 µl NaAc (3 M, pH=5), 1 µl glycogen (Invitrogen, Cat. 10814010). After keeping at -80℃ for 4 h, sRNA was pelleted by centrifugation at 20,000 ×g for 30 min at 4℃. The pellets was washed with 80% ethanol for two times and dissolved in 7 µl of RNase-free H2O.
NEBNext® Small RNA library prep kit (E7300)

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Galaxy platform
adaptors were removed with Trimmomatic (Bolger et al., 2014)
sequencing quality was accessed with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/)
Filtered reads (> 17 nucleotides) were mapped to the Arabidopsis genome (TAIR10) with with Bowtie2 (Langmead and Salzberg, 2012).
Reads were counted with featureCounts (Liao et al., 2014) with the help of the gene annotation in Araport11 (www.araport.org/data/araport11).
Genome_build: TAIR10
Supplementary_files_format_and_content: tab-delimited files include read count values for each sample

Submission date

Jun 09, 2021

Last update date

Jun 01, 2024

Contact name

Tatjana Kleine

E-mail(s)

tatjana.kleine@lmu.de

Organization name

Ludwig-Maximilians-Universität München

Department

Biologie I, Botanik