RNA-seq profiling for N. salina CCMP1776 under high salt and nitrogen limitation, RNA-seq, high salt and nitrogen limitation

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high salt and nitrogen limitation

Transcriptomic data for N. salina CCMP 1776 that had not been previously published, were based on four independent repeats of RNA-seq for normal, nitrogen (N) limitation and high salt stress conditions. The quality of raw reads was evaluated by SolexaQA software v3.1.5 (http://solexaqa.sourceforge.net/) (Cox et al., 2010) and cleaned reads were extracted by removing low-quality (phred < 20) reads from the dataset. The cleaned reads were mapped to the transcripts with the bowtie2 (v2.1.0) software (Langmead et al., 2009). The number of mapped reads for each transcript was calculated and normalized with DESeq package in R (Anders and Huber, 2010). The read counts of all genes were published through the expression menu and genome browse menu on "Nannochloropsis WebDB" (http://web.seeders.co.kr/NSK2019/), and now incorporated into NanDeSyn (http://nandesyn.single-cell.cn/organism/5).

Raw sequencing reads can be obtained by requesting to Byeong-ryool Jeong (bjeong@unist.ac.kr).

 

Cultivation and RNA extraction

N. salina WT and NsbHLH2 transformants were cultivated under normal, N limitation, and osmotic stress condition to isolate RNA as described in [1, 2]. F2N medium was used for normal condition. F2N media with 75 mg/L NaNO₃ 50 g/L sea salt were used for N limitation and osmotic stress condition, respectively. The culture conditions were followings: 25°C, 120 rpm, under 120 µmol photons/m²/s of fluorescent light, and 0.5 vvm air containing 2% CO₂. The samples were obtained at 4, 8, and 12 day to extract RNA. After cell harvest at the mid-exponential phase, 200 mg of wet biomass was used to obtain total RNA. All analysis was performed by quadruplicate.

 

RNA-seq data analyses

For pre-processing of short reads, DynamicTrim and Length sort program of SlexaQA (v.1.13) package was employed [3]. Bad quality bases of both ends of short reads were removed according to phred score by DynamicTrim. Subsequently, short reads which were considerably refined by DynamicTrim were removed by LengthSort. Finally, cleaned reads which have a phred score of more than 13 and a short read length of more than 25 bp were selected. De novo transcriptome assembly was conducted using Cufflink (v2.1.1). To form the locus transcript which is not assembled by Cufflink, Velvet (v1.2.08) [4] and Oases (v0.2.08) [5] program was additionally used. By CD-HIT, overlapped transcripts were removed. Basically, locus transcripts were selected by Cufflink, and the trasncripts which were not generated well by Cufflink were selected by Velvet & Oases. 

 

Reference

1.        Kang, N.K., et al., Effects of overexpression of a bHLH transcription factor on biomass and lipid production in Nannochloropsis salina. Biotechnol Biofuels, 2015. 8: p. 200.

2.        Kwon, S., et al., Enhancement of biomass and lipid productivity by overexpression of a bZIP transcription factor in Nannochloropsis salina. Biotechnol Bioeng, 2018. 115(2): p. 331-340.

3.        Cox, M.P., D.A. Peterson, and P.J. Biggs, SolexaQA: At-a-glance quality assessment of Illumina second-generation sequencing data. BMC Bioinformatics, 2010. 11: p. 485.

4.        Zerbino, D.R. and E. Birney, Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res, 2008. 18(5): p. 821-9.

5.        Schulz, M.H., et al., Oases: robust de novo RNA-seq assembly across the dynamic range of expression levels. Bioinformatics, 2012. 28(8): p. 1086-92.