|
| Status |
Public on Sep 14, 2023 |
| Title |
PolyA RNA ONS Rep 2 |
| Sample type |
SRA |
| |
|
| Source name |
Frond tissue
|
| Organism |
Lemna gibba |
| Characteristics |
nitrogen source: organic
tissue: frond
|
| Treatment protocol |
The inorganic SH medium contains ammonium phosphate monobasic (1.3 mM) and potassium nitrate (12.4 mM) as nitrogen sources. For organic nitrogen growth medium these inorganic nitrogen containing salts were replaced by equal concentration of potassium phosphate monobasic and potassium chloride, respectively. Gln was added at 6.85 mM in order to provide nitrogen at the same concentration as in the SH medium.
|
| Growth protocol |
Axenic cultures of Lemna gibba G3 7742a were obtained from Rutgers University Duckweed Stock Cooperative (www.ruduckweed.org, ID 7742a/DWC131) and were cultured in SH medium (Sigma S6765, half as manufacturer’s recommended concentration, 1.6g/L) (Schenk and Hildebrandt 1972) with 5 g/l glucose (pH 5.7). Fronds were cultured axenically in T-75 cell culture flask with vented caps filled with 100 ml of the culture medium, at 24°C, under continuous fluorescent light (100 µmol*m^-2*s^-1).
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Fronds grown on inorganic nitrogen or on organic nitrogen medium were harvested and immediately frozen in liquid nitrogen. For each condition three biological replicates were analyzed. Total RNA was extracted by grinding 200 mg frozen tissue to a fine powder in liquid nitrogen with a mortar and pestle, followed by extraction with TRIzol Reagent (Invitrogen) and ethanol precipitation. Total RNA was resuspended and treated with RQ1 RNase-free DNase (Promega) on an RNA Clean & Concentrator-5 column (Zymo). The purity and concentration of RNA were determined by a NanoDrop Nd-1000 spectrophotometer (Thermo Scientific) and a Qubit fluorometer (Qiagen).
Polyadenylated RNA was enriched from total RNA with the Dynabeads mRNA Purification Kit (Life Technologies). Indexed, directional RNA-seq libraries were prepared with ScriptSeq v2 reagents (Epicentre) from 40-50 ng of polyA-selected RNA according to the manufacturer’s instructions. Libraries were sequenced on an Illumina HiSeq 2000 instrument generating 101bp paired-end reads.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Reads from each sample were mapped against the 21,830 predicted transcripts of the Lemna gibba draft genome v0.5.1 (www.lemna.org; genomevolution.org/coge/GenomeInfo.pl?gid=25249) using Bowtie (Langmead and Salzberg 2012).
Differential expression analysis for the two conditions (INS, ONS) was performed using the DESeq package (www.bioconductor.org, v1.20.0, (Anders and Huber 2010). This procedure included normalization of RNA-seq read count data (estimateSizeFactors, using default parameters) and the negative binominal test as implemented in DESeq. Significant differential expressed genes were defined for an adjusted p-value ≤ 5% (Benjamini-Hochberg anjustment for multiple testing) and a more than four-fold change in gene expression between conditions.
Genome_build: Lemna gibba 7742a draft v0.5.1
Supplementary_files_format_and_content: DESeq differential expression analysis full tabular output
|
| |
|
| Submission date |
Jun 30, 2020 |
| Last update date |
Sep 14, 2023 |
| Contact name |
Robert A Martienssen |
| E-mail(s) |
martiens@cshl.edu
|
| Organization name |
Cold Spring Harbor Laboratory
|
| Department |
Delbruck Bldg.
|
| Lab |
Martienssen
|
| Street address |
1 Bungtown Rd
|
| City |
Cold Spring Harbor |
| State/province |
NY |
| ZIP/Postal code |
11724 |
| Country |
USA |
| |
|
| Platform ID |
GPL28798 |
| Series (1) |
| GSE153602 |
Integrated metabolic and transcriptomic analysis of the duckweed Lemna gibba growing under photomixotrophic conditions: Metabolic adjustments to different nitrogen sources |
|
| Relations |
| BioSample |
SAMN15411685 |
| SRA |
SRX8644578 |
