Epicenter ribo zero rrna removal kit

The sequencing in the present study belongs to the same batch as that in a recent study [66 (link)] and is, therefore, methodologically identical. Total RNA was extracted from cow milk MECs using the TRIzol method. RNA degradation and contamination were monitored on 1% agarose gels. RNA purity was checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 System (Agilent Technologies, CA, USA). The 260/280 ratio of all samples ranged from 1.70 to 1.90, and the RNA Integrity Index (RIN) was ≥ 8.00. Sample RNA for circRNA sequencing was stripped of ribosomal RNA (Epicenter Ribozero™ rRNA Removal Kit, Epicentre, USA), and linear RNA was digested with RNase R (Epicentre, USA). Sequencing libraries were prepared according to the manufacturer’s instructions for the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA). After passing the library inspection, Illumina PE150 sequencing was performed. After filtering the raw data, the obtained clean reads were aligned with the downloaded reference genome (https://bovinegenome.elsiklab.missouri.edu/downloads/ARS-UCD1.2) using the Bowtie2 software (v2.2.8).

A total of 3 μg of RNA per sample was used as input material for the RNA sample preparations of lncRNA sequencing. First, ribosomal RNA was removed using a Epicenter Ribo-zero™ rRNA Removal Kit (Epicenter, USA), and rRNA-free residue was washed by ethanol precipitation. Subsequently, sequencing libraries were generated using an rRNA-depleted RNA by NEBNext® Ultra™ Directional RNA Library Prep kit for Illumina® (NEB, USA) following the manufacturer’s recommendations. Sequencing libraries of small RNA were generated using an NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (NEB, USA) following manufacturer’s recommendations, and index codes were added to the attribute sequences in each sample [61 (link)].

Total RNA was isolated from nine ginger rhizome samples using an RNeasy Plant Mini Kit (QIAGEN, Germany). The quality and integrity of the total RNA were evaluated using Nanodrop, Qubit 2.0, (Invitrogen, USA), and an Agilent Bioanalyzer 2100 System (Agilent Technologies, USA). The Epicenter Ribo-Zero™ rRNA Removal Kit (Epicenter, Madison, WI, USA) was used to remove ribosomal RNA. Three micrograms of rRNA-depleted RNA were used to construct a sequencing library using the NEBNext Ultra Directional RNA Library Prep Kit (NEB, USA) according to the manufacturer’s instructions. Nine libraries were sequenced on the Illumina PE150 platform. The sequencing results were deposited in the National Center for Biotechnology Information database under accession number (PRJNA870703), which was released since November 12^th, 2022.

Total RNA was divided into two samples after preparation. In one sample, ribosomal RNA was removed by Epicenter Ribo-zero™ rRNA Removal Kit (Epicenter, Madison, WI, USA), and residual RNAs were cleaned by ethanol precipitation. The sequencing libraries were generated using rRNA-depleted RNA with a NEB Next® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA). The constructed libraries were evaluated on an Agilent Bioanalyzer 2100 system (12 (link)). RNA integrity was checked by microcapillary electrophoresis using an Agilent 2100 Bioanalyzer with an RNA 6000 Nanochip kit (Agilent Technologies, Germany) (15 (link)). Sequencing was then performed using a paired-end 125-cycle rapid run on an Illumina HiSeq2500 (Illumina Inc., San Diego, CA, USA). Low-quality reads were removed, and the clean reads were filtered from the raw reads and mapped to the porcine reference genome (Sus scrofa v10.2). The mapped reads for each sample were independently assembled using Cufflinks (v2.1.1).

Total RNAs were isolated from thoracic aorta from SHR and WKY rats, a total amount of 5 μg RNA per sample was used as input material for the RNA sample preparations using TRIzol reagent (Invitrogen, USA). Furthermore, RNA purity was checked using the NanoPhotometer®spectrophotometer (IMPLEN, CA, USA) and RNA integrity was evaluated using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Aglient Technologies, CA, USA). Additionally, ribosomal RNA was depleted from total RNA using the Epicenter Ribozero™ rRNA Removal Kit (Epicenter, USA), and rRNA free residue was cleaned up by ethanol precipitation. Subsequently, the linear RNA was digested with 3U of RNase R (Epicenter, USA) per μg of RNA. The sequencing libraries were generated by NEBNext®Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations by Shanghai Genechem Co., Ltd., Shanghai, China. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer's instructions. After cluster generation, the libraries were sequenced on an Illumina platform and 150 bp paired-end reads were generated.

Total tissue RNA was extracted using TRIzol reagent (Invitrogen™, Carlsbad, CA, USA) according to the manufacturer's manual, and the extracted RNA was tested for quality and integrity (Agilent Technologies, CA, USA). Next, rRNA was removed using the Epicenter-Ribo-zero rRNA Removal Kit (Epicentre, USA), and rRNA-free residues were removed by ethanol precipitation. Then, sequencing libraries were generated using NEBNext Ultra Directed RNA Library Preparation Kit for Illumina (NEB, USA). The library was sequenced at Novogene (Beijing, China) using the Illumina Hiseq 4000 base platform, and 150 bp paired-end reads were generated.

Leaf samples were used to extract total RNAs and generate a complementary (c)DNA library of sRNAs. sRNA-seq was carried out using an Illumina HiSeq^TM 2000 system (SinoGenoMax, Beijing, China), as reported previously (Fan et al., 2016 (link)). “Clean” data were obtained by removing sequences <18 nucleotides (nt) or >30 nt, low-quality tags, poly-A tags, and N tags from raw reads. Sequences of potential viruses were identified by analyses of clean data using VirusDetect¹ (Zheng et al., 2017a (link)). After preliminary identification of a novel emaravirus by sRNA-seq, the same sample was also used to identify other potential sequences of the newly identified virus by RNA-seq, and also to obtain longer assembled sequences. For RNA-seq, the Epicenter Ribo-Zero rRNA Removal Kit (Epicenter, Madison, WI, United States) was used to remove ribosomal RNA from extracts of total RNA. The ribosomal RNA-depleted RNA sample was then used to construct a cDNA library using a TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA, United States), which was sequenced on an Illumina HiSeq 4000 platform with a paired-end 150-bp setup (Biomarker Biology Technology). Reads mapping to the grapevine genome (PN40024 assembly 12X) were filtered out by hierarchical indexing using hisat software (Kim et al., 2015 (link)). Unmapped reads were used for de novo assembly and Blast analysis embedded in VirusDetect.