Zymo seq ribofree total rna library prep kit

Total RNA samples were subjected to Ribosomal RNA depletion, then used for library preparation using Zymo-Seq RiboFree Total RNA Library Prep Kit (Zymo Research, USA). Sequencing was performed on the Illumina Hiseq 2000/2500 sequencer by Shanghai Biotechnology Corporation (China). The low-quality reads (including reads shorter than 20 bp, low-quality scores (<20), and adapter sequences) were filtered out from the raw reads obtained. The mRNA sequences of S. luteus in the raw reads were filtered out by aligning the reads to the reference sequence of the S. luteus strain UH-Slu-Lm8-n1 (https://www.ncbi.nlm.nih.gov/datasets/taxonomy/930992/). The clean reads were de novo assembled into Primary UniGenes, using the scaffolding contig algorithm of CLC Genomics Workbench software (version: 6.0.4). The resulting Primary UniGenes were then used to construct the first_contig and the second_contig (with stricter splicing parameters) using CAP3 EST. The contigs obtained were subjected to BLASTx analysis on National Center for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov/) Non-Redundant Protein database.

Ribosomal RNA depletion, library preparations, and Illumina sequencing were performed by Shanghai Biotechnology Corporation (China). Zymo-Seq RiboFree Total RNA Library Prep Kit (Zymo Research, USA) was used for ribosomal RNA depletion and library preparations. Illumina sequencing (Illumina NovaSeq 6000) was carried out by Shanghai Biotechnology Corporation (China).The filtered data was spliced together from scratch, and the resulting sequences were then de-duplicated. Subsequently, the virus sequences have been annotated using Diamond software (version 0.9.21.122) and the National Center for Biotechnology Information (NCBI) Non-Redundant Protein database.¹

Samples were submitted to Zymo Research (Irvine, CA, USA) for total mRNA extraction, cDNA library preparation, and RNA sequencing. Total RNA-Seq libraries for pigs were constructed from 250 ng of total RNA. To remove rRNA, a method described by Bogdanova et al., 2011 was followed. Libraries were prepared using the Zymo-Seq RiboFree Total RNA Library Prep Kit (Cat # R3000) according to the manufacturer’s instructions (Zymo-Research, 2022 ). The RNA-Seq libraries were sequenced on an Illumina NovaSeq to a sequencing depth of at least 30 million read pairs (150 bp paired-end sequencing) per sample.

Metatranscriptome sequencing of R. zeae strain D40 was carried out by Shanghai Biotechnology Corporation using an Illumina NovaSeq 6000 platform for paired-end sequencing. Sequencing libraries were established from rRNA-depleted total RNA samples of strain D40 using a Zymo-Seq RiboFree Total RNA Library Prep Kit (Zymo Research, California, CA, USA). Library quality assessment was determined on a Qubit^® 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) and Agilent Technologies 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Low-quality reads and fungi mRNA sequences were filtered out from the resulting raw reads, and thus high-quality clean reads were obtained. A de novo assembly of the primary unigenes was constructed using CLC Genomics Workbench version 6.0.4 software. Final unigenes were assembled using CAP3 EST software. The resulting final unigenes were queried against the National Center for the Biotechnology Information (NCBI) non-redundant (NR) database and aligned using BLASTx to obtain homologous sequences of mycoviruses. The unmatched contigs, both with size of nucleotides over 1.5 kb and number of reads over 1000, were kept [26 (link)].

The strains ID40 and ID40∆ygfB were used to perform RNA sequencing and differential gene expression analysis (ID40 vs ID40∆ygfB). The strains were subcultivated for 3 h in 5 ml LB medium. A total of four independent replicates per strain were used in the sequencing and analysis. RNA was isolated using the Quick-RNA™Fungal/Bacterial MiniprepKit (Zymo Research) according to manufacturer´s instruction. Subsequently, 15 µg RNA in 50 µl water was digested with 10 U DNAse I (Roche). The quality of the RNA was controlled by determination of the RNA Integrity Index using Agilent BioAnalyzer High Sensitivity DNA Assays. Successful depletion of DNA was controlled by qPCR and RT-qPCR for the rpoS and the ygfB genes. Next, the Zymo-Seq RiboFree total RNA Library Prep Kit (Zymo Research) was used to deplete ribosomal RNA and prepare samples for sequencing. For this step, 2 µg RNA per sample were used. Sequencing was performed with Illumina NextSeq500 (2×75 bp, MidOutput Flowcell). Mapping of sequencing reads and counting was performed using the subread package in R and the ID40 genome as a reference (https://www.ebi.ac.uk/ena/browser/view/LR700248)^{37 (link)}. Differential gene expression analysis was performed using DeSeq2^{38 (link)}.

For RNA extraction, cells were grown in CYE liquid medium at 32°C with shaking at 160rpm in the dark. Then, 1ml of cells at OD₆₀₀ 1.0 were harvested by centrifuging at 8000rpm for 5min. Cells pellets were frozen by soaking in liquid nitrogen for 2min. RNA extraction was carried out using the Maxwell miRNA Tissue Kit (Promega) following the manufacturer’s instructions. Removal of rRNA and cDNA synthesis were carried out using Zymo-Seq RiboFree total RNA library Prep Kit (Zymo Research) following the manufacturer’s instructions. The cDNA from three biological triplicates of each sample was used for sequencing using the Illumina system (75bp paired-end reads) at the IMM Transcriptomic Platform. Sequence reads were pre-processed to remove low-quality bases and also cut the adaptors using Galaxy. Next, remaining reads were subsequently mapped to the M. xanthus DK1622 genome with the default parameters and using the pair-end strategy. Htseq-count were used to count the reads for each gene. SARtools and DEseq2 were used to analyze differential gene expression between wt and mutants. Differential expression was characterized by filtering with FDR < 0.01 and p < 0.001. RNAseq experiments were performed in three biological replicates.