Epicenter ribo zero gold kit

As previously described (Li Y. C. et al., 2020 (link)), total RNA was extracted using TRIzol Reagent (Invitrogen), and the RNA concentration was assessed with an RNA 6000 Nano LabChip Kit (Agilent). An Epicenter Ribo-Zero Gold Kit (Illumina) was used to remove ribosomal RNA from 10 μg of total RNA. The RNA fraction was isolated after purification, and then a cDNA library was created according to the instructions of an mRNA-Seq sample preparation kit (Illumina). Libraries with cDNA target fragments of 250–350 bp were selected. HiSeq 4000 (Illumina) was used for paired-end sequencing, and subsequent sequencing experiments and bioinformatics analyses were performed at LC Bio (China).

For circRNAs and mRNA, the total RNA samples were treated with an Epicenter Ribo‐Zero Gold Kit (Illumina) to remove rRNA before constructing RNA‐seq libraries. The samples were fragmented and then synthesized as first‐ and second‐strand complementary DNA (cDNA) with random hexamer primers, dNTPs and DNA polymerase I using a PrimeScript RT reagent Kit (TaKaRa). The cDNA fragments were treated with T4 DNA polymerase to repair the ends and Klenow DNA polymerase to add ‐A and adapters at the 3′ end of the DNA fragments. The cDNA products were purified with AMPure XP beads and then subjected to PCR amplification. Then, the cleaved RNA fragments were reverse‐transcribed to create the final cDNA library in accordance with the mRNA‐Seq sample preparation kit protocol (Illumina). Then, we performed paired‐end sequencing on a HiSeq 4000 sequencing system (Illumina) following the manufacturer's recommended protocol.
For miRNAs, approximately 1 μg of total RNA was used to prepare a small RNA library according to the TruSeq Small RNA Sample Prep Kit protocol (Illumina). Then, we performed single‐end sequencing (1 × 50 bp) on an Illumina Hiseq 2500 sequencing system following the manufacturer's recommended protocol.

Ribosomal RNA was depleted as per the instructions provided in the Epicenter Ribo-Zero Gold Kit (Illumina, San Diego, USA). The cDNA libraries were created following fragmentation of the poly(A)−/(A) + RNA fractions, reverse-transcription. Paired-end sequencing was conducted on an Illumina HiSeq 4000 (LC-Bio, China). The reads with adaptor contamination, low quality and undetermined bases were removed, followed by quality verification using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). We used Bowtie 2 [60 (link)] and TopHat2 [61 (link)] to map reads to the genome of Nipponbare rice Reference Genome (IRGSP build 5.0). The mapped reads of each sample were assembled using StringTie [62 (link)]. After the final transcriptome was generated, Ballgown [63 (link)] was used to estimate the expression levels of all transcripts. The raw data was available at NCBI Gene Expression Omnibus (GEO) repository with Accession Number GSE142323.

Total RNA was extracted using TRIzol reagent (Invitrogen, CA, United States) according to the manufacturer’s instructions. The RNA quantity and purity were determined using a NanoDrop ND-1000 (NanoDrop, Wilmington, DE, United States) and Bioanalyzer 2100 (Agilent, CA, United States). A small RNA library was prepared using approximately 1 µg of total RNA according to the TruSeq Small RNA Sample Prep Kit protocol (Illumina, San Diego, CA, United States). We then performed single-end sequencing (36 or 50 bp) on an Illumina HiSeq 2500 system (Illumina, San Diego, CA, United States) at LC-Bio (Hangzhou, China).
Ribosomal RNA was removed from approximately 2 µg of total RNA using the Epicenter Ribo-Zero Gold Kit (Illumina, San Diego, CA, United States) according to the manufacturer’s instructions. The purified ribosomal RNA was fragmented into small pieces using divalent cations at elevated temperatures. The cleaved RNA fragments were then reverse-transcribed to generate the final cDNA library in accordance with the mRNA-Seq sample preparation kit protocol (Invitrogen, CA, United States). The final cDNA library had an average insert size of 300 ± 50 bp. We then performed 2 × 150-bp paired-end sequencing (PE150) using an Illumina NovaSeq™ 6000 system (LC-Bio Technology Co., Ltd., Hangzhou, China).

Total RNA was extracted using TRIzol reagent (Invitrogen, CA, United States) following the manufacturer’s procedure. The 6000 Nano LabChip Kit (Agilent, CA, United States) with the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, United States) was used to assess RNA integrity. Approximately 1 μg of total RNA with an RNA integrity number (RIN) > 7.0 was used to prepare a small RNA library according to the protocol of a TruSeq Small RNA Sample Prep Kit (Illumina, San Diego, United States). Then, we performed single-end sequencing (36 bp or 50 bp) on an Illumina HiSeq 2500. Approximately 10 μg of total RNA representing a specific adipose type was used to deplete ribosomal RNA with an Epicenter Ribo-Zero Gold Kit (Illumina). Following purification, the poly(A)- or poly(A)+ RNA fractions were fragmented into small pieces using divalent cations under elevated temperature. Then, the cleaved RNA fragments were reverse-transcribed to create the final cDNA library in accordance with the protocol for a mRNA-Seq sample preparation kit (Illumina), and the average insert size for the paired-end libraries was 300 bp (±50 bp). Then, we performed paired-end sequencing on an Illumina HiSeq 4000 (lc-bio, China) following the vendor’s recommended protocol.

EPC cells were seeded at a density of 2 × 10⁴ cells per well in 96-well plates, whereas cells were cultured at a density of 5 × 10⁴ cells per well. EPC cells were infected with 0.1 multiplicity of infection (MOI) of IHNV for 1 h, then washed with PBS and cultured in MEM containing 2% FBS at 15 °C and 5% CO₂ to establish the IHNV infected model. Cells in the IHNV infected group (IH group) and IHNV uninfected group (NC group) were obtained after 72 h post-infection (pi). Total RNA was extracted from the cells using a PrimeScript RT reagent kit with a DNA Eraser (TaKaRa, Shiga, Japan) kit according to the manufacturer’s instructions. The quality of RNA was determined using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The Epicenter Ribo-Zero Gold kit (Illumina, San Diego, CA, USA) was used to eliminate ribosomal RNA (rRNA) from total RNA, and reverse transcription of the RNA was conducted to obtain cDNA templates.