PRJNA157867, RNA-seq, N+/N-, PRJNA157867


Overview
Analysis NamePRJNA157867
Organism NameNannochloropsis oceanica (N. oceanica CCMP1779)
Methodnf-core/rnaseq (nf-core/rnaseq v1.2)
SourceRNA-seq, N+/N-, PRJNA157867
Date performed2019-07-02

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RNA sequencing data from N-replete and N-depleted

This dataset was downloaded from NCBI bioproject: PRJNA157867 (GEO: GSE36959).

Below detailed description was retreived from Reference: Vieler A et al., "Genome, functional gene annotation, and nuclear transformation of the heterokont oleaginous alga Nannochloropsis oceanica CCMP1779.", PLoS Genet, 2012;8(11):e1003064.

 

Strains and growth conditions

The Nannochloropsis sp. strain used was CCMP1779, available from The Provasoli-Guillard National Center for Culture of Marine Phytoplankton (https://ncma.bigelow.org/). The cells were grown in liquid cultures under continuous light (∼80 µmole photons m−2 s−1). For N-replete growth, f/2 medium with 2.5 mM nitrate (f/2+N) was used [25]. For nitrogen-deprived experiments, N deprivation was applied by growth in f/2+N to 1×107 cells mL−1, followed by transfer to f/2 without nitrogen source to 5×106 cells mL−1 for an additional 30 hours.

DNA and RNA preparation for sequencing and analysis

For preparation of nuclear DNA a 50 mL cell culture (OD750 = 0.4 to 0.5) was harvested by centrifugation (4,500× g, 5 min). The cell pellet was lysed in 2× cetyltrimethylammonium bromide (CTAB) buffer (2% CTAB, 100 mM Tris-HCl pH 8.0, 1.4 M NaCl, and 20 mM EDTA) and incubated at 60°C for 60 min. The lysate was mixed with 1 volume of phenol/chloroform and centrifuged (13,000× g min). Transferred the supernatant to a new tube and repeated this step at least once until there was no white interphase. The DNA was precipitated by 1 volume isopropanol and 70% ethanol. High molecular weight DNA was examined by DNA gel electrophoresis.

To generate material for RNA-sequencing, cells were grown in 200 ml f/2+N to 1×107 cells mL−1. The cultures were split in half and cells were collected by centrifugation (4,500× g, 5 min), with one pellet being resuspended in 200 mL f/2+N, and the other in 200 mL f/2-N. After 30 hours, the total RNA was isolated using TRIzol Reagent (Invitrogen) according to manufacturer's instructions. The RNA samples were cleaned up using RNeasy columns (Qiagen, http://www.qiagen.com) following the manufacturer's instruction.

Assessment of RNA quantity and quality

The evaluation of RNA quantity and quality was done spectrophotometrically by UV absorbance profile. Additional analysis was performed using an RNA 6000 Nano LabChip Kit for microcapillary electrophoresis (Agilent 2100 Bioanalyzer, http://www.home.agilent.com). This eukaryotic total RNA nano-assay generated information about RNA integrity through electropherograms, gel picture, and RIN value (RNA Integrity Number) [155].

Transcript assembly and differential expression analysis

De novo transcript assemblies were generated from 55 bp directional single-end Illumina reads of N-replete and N-depleted conditions (NCBI/GEO GSE36959) using Oases (http://www.ebi.ac.uk/~zerbino/oases/). First, Oases was run for k-mer lengths of 23, 25, 27, 29, 31, 33, 35, and 37, and the results were compiled. To identify a set of high confidence transcripts from the de novo assemblies, proteins from six sequenced heterokont genomes, including Ectocarpus siliculosus [6]Pythium ultimum [161]Phytophthora sojae [162]Phytophthora ramorum [162]Thalassiosira Pseudonana [64], and Phaeodactylum tricornutum[4], were aligned to the de novo transcripts and only those with significant matches to known proteins were kept. These transcripts with cross-genome matches were mapped back to the Illumina genome assemblies to evaluate genome assembly quality. In addition to de novotranscript assembly, we generated a genome-based transcript assembly.

Transcriptomic reads from N-replete and N-depleted conditions were separately mapped to the hybrid genome assembly using Tophat [160] (parameters: -I 10 –I 3000 –library-type fr-unstranded –g 1). The mapped reads were assembled into transcripts using Cufflinks [163] (-I 3000 –library-type fr-secondstrand) and a set of transcripts was generated for each condition.

Biomaterial Browser
The following browser provides a list of biomaterials associated with this analysis.
Biomaterial NameOrganismBiomaterial ProviderTreatment
GSM907185Nannochloropsis oceanica (N. oceanica CCMP1779)CCMP1779Nitrogen replete
GSM907186Nannochloropsis oceanica (N. oceanica CCMP1779)CCMP1779Nitrogen depleted

Feature Expression

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