Computational protocol: Comparing photosynthetic characteristics of Isoetes sinensis Palmer under submerged and terrestrial conditions

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Protocol publication

[…] We applied RNA-sequencing technology to obtain photosynthetic genes in Isoetes. Briefly, juvenile leaves were harvested under TC and SC, respectively. Then the leaves were immediately frozen in liquid nitrogen and stored at –80°C prior to RNA extraction. Total RNA was extracted using Trizol (Invitrogen Inc., USA) and residual DNA was further removed with RNase-free DNase I (Takara, Da Lian, China) according to the manufacturers’ instruction. The RNA quality and quantity were verified using 2100 Bioanalyzer RNA Nanochip (Aligent, CA, USA) and ND-1000 Nanodrop Spectrophotometer (Thermo scientific, DE, USA), respectively. Equal amounts of total RNA were mixed from three independent extractions to a single combined sample. The mRNA was purified by oligo(dT) magnetic beads, fragmented to about 200 bp, and further synthesized into first-strand cDNA using random hexamer primers and reverse transcriptase. Then second-strand cDNA was synthesized using RHase H (New England Biolabs Inc., Ipswich, MA, USA) and DNA polymerase (Invitrogen, Carlsbad, CA, USA). We further repaired the end fragments and ligated them with sequencing adaptors. Appropriate fragment ranges for PCR application (200±25bp) were selected by agarose gel electrophoresis and purified using a QiaQuick PCR extraction kit (Qiagen, USA). Finally, the cDNA libraries were sequenced on an Illumina Hiseq 2000 platform to generate 100 pair-end raw reads, according to the manufacturers’ recommendation (Illumina Inc., San Diego, CA, USA).The raw reads initially conducted a general quality control analysis using FastQC. The filtering thresholds included removing adaptors, ambiguous nucleotide reads with N, polymerase chain reaction artifacts, and low quality bases with an average Phred score less than 20. After filtering and trimming the contaminations and bad quality sequences, all clean reads were merged and further de novo assembled into unigenes using Trinity program, setting k-means length to 25. All unigenes were further searched using BLASTx against the non-redundant database with an E-value cutoff of 10−5. For the sequences failed to be searched in the database, we used GetORF software to predict their orientations and underlying protein coding regions. [...] Approximately 0.1 g harvested materials were immediately frozen by liquid nitrogen in a cold mortar. Total RNA was extracted using RNAiso Plus (Takara, Da Lian, China), according to the manufacturers’ instruction. The extracted RNA was incubated using 1 μL RNase-free DNase I (Promega, Madison, WI, USA) for 15 min at 37 °C to eliminate genomic DNA. Then 1μg total RNA was reverse transcribed into single-strand cDNA using PrimerscriptTM One Step RT-PCR Kit Ver.2 following the manufacturers’ protocol (Takara, Da Lian, China). Gene-specific primers were designed using a free online primer design tool (http://primer3plus.com/cgi-bin/dev/primer3plus.cgi). The qPCR analysis was performed on a CFX96 Real-time PCR system (Bio-Rad, Hercules, USA). The cDNA initially was diluted ten-fold and used as templates in the qPCR tests. The reaction mixtures (25 μL) included 0.25 pmol forward and reverse primers, 12.5 μL 2 × SYBR premix (Takara, Da Lian, China), 2.5 μL diluted cDNA, and 7.0 μL sterile water. The PCR program was an initial denaturation step of 3 min at 95 °C, followed by 40 cycles of 95 °C for 30 s, annealing temperatures for 15 s and finally 72 °C for 30 s. Specific annealing temperature and related information for the photosynthetic genes are listed in . The relative expression levels were calculated using 2−△△Ct method, and normalized to the geometric average of Ct values with the actin gene as an internal control. All experiments and analysis were performed in triplicate. […]

Pipeline specifications

Software tools FastQC, Trinity, BLASTX, EMBOSS, Primer3
Applications RNA-seq analysis, qPCR