Qubit rna assay kit on qubit 2.0 fluorometer

Extraction of total RNA was conducted out of ovules by utilizing a Total RNA Extraction kit (Aidlab, Beijing, China) in line with the manufacturer’s protocol. 1% agarose gels was performed for inspection of the RNA contamination and degradation. NanoPhotometer® spectrophotometer (IMPLEN, CA, USA) was used to examine the RNA purity. Qubit® RNA Assay Kit on Qubit® 2.0 Fluorometer (Life Technologies, CA, USA) was utilized for detecting the RNA concentration. Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA) to test the RNA integrity.
For every sample, 3 μg total RNA was applied for the construction of Small RNA library. With the manufacturer’s instruction, the NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (NEB, USA.) was employed for generating of the sequencing library. Lastly, Illumina HiSeq 2500/2000 platform was used to sequence the library, thereby generating 50 bp single-end reads.

Total RNA of all prokaryotes was isolated using RNAiso Plus reagent (TaKaRa, Dalian, China) after a lysozyme digestion procedure. Fungal RNA was extracted as described by Mannan et al. [24 (link)]. Total nucleic acid (NA) was extracted from 0.5 g of salt-marsh sediments as recommended by Lüdemann et al. [25 (link)]. Isolation of total NA from the remaining five samples was conducted using a mirVana RNA Isolation Kit (Ambion, Austin, TX) according to the manufacturer’s instructions after a glass beads vortex step.
The total RNA or NA was visualized in a 1% (w/v) agarose gel after electrophoresis to assess the sample integrity. The bands containing SSU rRNA of microbial isolates and Pa sediments were excised from the agarose gel, and SSU rRNA was then purified from the gel using a Qiaquick Gel Extraction Kit (Qiagen, Hilden, Germany). Electrophoresis was conducted to assess the integrity of enriched SSU rRNA, and a BioAnalyzer 2100 (Agilent Technologies, Palo Alto, CA) was used to check for any contamination.
Total NA from all samples was stored at -80°C and RNA samples were quantified using a Qubit RNA Assay Kit on Qubit 2.0 fluorometer (Life Technologies, Carlsbad, CA) before the preparation of RNA-seq libraries.

Total RNA was isolated by TRIzol reagent and genomic DNA was removed by DNase I (Tiangen Biotech Co., Ltd., Beijing, China). NanoPhotometer^® (IMPLEN, Los Angeles County, CA, USA), Qubit^® RNA Assay Kit on Qubit^® 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA Nano 6000 Assay Kit on Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA) were used to test RNA purity, concentration and integrity, respectively. Only qualified samples were used for library preparation. Sequencing libraries were generated using the TruseqTM RNA Sample Prep Kit (Illumina, San Diego, CA, USA) as recommended by the manufacturer of the instrument. Sequencing was performed using the Illumina NovaSeq 6000 platform to produce 150 bp paired-end reads. The original sequencing data can be found in the Sequence Read Archive database under accession number PRJNA903782.