Truseq stranded mrna preparation kit

Total RNA was extracted from PFC samples using the RNeasy Lipid Tissue Mini Kit (QIAGEN, Madrid, Spain), and total RNA concentration and purity were assessed with a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Spain). For quality check, the OD 260/280 ratio was determined to be between 1.87 and 2.0. RNA integrity number (RIN), was determined using the Bioanalyzer-2011 equipment (Agilent Technologies, Santa Clara CA, USA). To guarantee their preservation, RNA samples were treated with RNAstable (Sigma-Aldrich, Spain), and shipped at ambient temperature to DNA-link Inc. sequencing laboratory (Seoul, Korea) to perform high throughput sequencing using a Novaseq 6000 sequencer (Illumina, San Diego, CA, USA). All these procedures were conducted according to the respective manufacturers´ protocols.
Individual libraries for each of the analyzed bovines (N = 16) were processed using the TruSeq Stranded mRNA Preparation kit (Illumina, San Diego, CA, USA) according to manufacturer´s instructions. All samples were sequenced in the same flowcell and lane following a pair-end protocol (2 × 100 bp). Individual reads were de-multiplexed using the CASAVA pipeline (Illumina v1.8.2), obtaining the FASTQ files used for downstream bioinformatics analysis.

Total RNA was extracted from empty vector control and FCN3-virus-transduced A549 cells, and mRNA libraries were constructed by the TruSeq Stranded mRNA Preparation kit (Illumina) according to the manufacturer’s instructions. RNA-seq was performed with Illumina NovaSeq 6000 sequencing platform for 101-mer paired-end reads (DNA Link Inc., Seoul, Korea). Sequencing reads were aligned to the reference human genome (hg19) using STAR alignment program (version 2.6.1d)^{22 (link)} after trimming adapter sequences and discarding low-quality reads using Fastx_toolkit (version 0.0.13). Transcriptome abundance was estimated with RSEM (version 1.3.3)^{16 (link)}. DEGs were identified using DESeq2^{23 (link)} with the FDR threshold of 0.01. Gene set analyses for DEGs were performed with EnrichR^{24 (link)} using KEGG pathways and gene ontology (GO) terms. The RNA-seq data have been deposited in the Gene Express Omnibus (GEO) database [GEO: GSE160946].

Total RNAs were extracted from the livers as described above. RNA-seq libraries were generated using the Illumina TruSeq Stranded mRNA preparation kit according to the manufacturer’s instructions (Illumina, CA, USA). Sequencing experiments were performed with NovaSeq 6000 system (Illumina; Single End 100 bp). The obtained reads were mapped to the mouse genome mm10 using Illumina Eland with the default parameter setting. The obtained gene list with reads per million per kilobase (RPKM) scores were shown in Supplementary Data 2. RPKM scores from four replicates were averaged, and the ratio between 4T1-bearing and sham-operated conditions was calculated in WT and Nnmt KO. The ratio between sham-treated WT and Nnmt KO was also calculated. The obtained ratios were used to sort genes to find candidate differentially expressed genes (DEGs). DEGs were further subjected to gene ontology analyses using g:Profier^{44 (link)}.

RNA was extracted from variably treated A549 cells, and mRNA libraries were prepared using the TruSeq Stranded mRNA Preparation kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. RNA sequencing (RNA-Seq) was performed with Illumina HiSeq2500 sequencing platform for 101-mer paired-end reads. TrimGalore (version 0.6.7) was used for trimming adapter sequences and discarding low-quality reads. The reads were mapped against the human reference genome (GRCh38/hg38) using the STAR alignment program (version 2.7.9a) [20 (link)]. Gene expression matrix was generated using FeatureCount function in Rsubread version 2.6.4 [21 (link)]. Differentially expressed genes (DEGs) were identified using DESeq2 with the adjusted p-value < 0.05 and |fold change| > 2 threshold [22 (link)]. Gene set analyses of DEGs for pathways and gene ontology (GO) terms and Gene Set Enrichment Analysis (GSEA) were performed using clusterProfiler version 4.0.4, an R package for interpretation of omics data [23 (link)]. The RNAseq data have been deposited in the Gene Expression Omnibus (GEO) database [GEO: GSE185306].

RNA extracted from viruses #1, #2 and #3 was processed following the protocol for the Illumina TruSeq Stranded mRNA Preparation Kit (Illumina), but without the polyA enrichment step. Briefly, approximately 100 ng of RNA was chemically fragmented and reverse-transcribed into cDNAs. Double strand cDNAs were adenylated at the 3’ends and individually indexed, followed by limited-cycle (15) amplification, and purification using Agencourt AMPure magentic beads (Beckman Coulter). After analyzing the cDNA libraries for size and quality using a BioAnalyser (Agilent Technologies), paired-end sequencing (100 x 2 cycles) of twelve multiplexed RNA samples per lane was carried out on an Illumina HiSeq2500 sequencer. HiSeq sequencing data generated for viruses #1, #2 and #3 and for the repeat HiSeq run for virus #2, are available under https://www.ncbi.nlm.nih.gov/sra/PRJNA525871 (files are named Virus_1_18C, Virus_2_19C, Virus_3_20C and Virus_2_HiSeq_rpt_11S)

Total RNAs were extracted from bone marrow cells as described above. The RNA quality was assessed by Agilent 2100 bioanalyzer using the RNA 6000 Nano Chip obtained from Agilent Technologies (Amstelveen, The Netherlands). RNA-seq libraries were generated using the Illumina TruSeq Stranded mRNA preparation kit according to the manufacturer’s instructions (Illumina, CA, USA). Sequencing experiments were performed with NovaSeq 6000 system (Illumina; Single End 100 bp). After quality control of the data using Fastp tool version 0.20.0^{45 (link)}, trimmed reads were mapped to mm10 by STAR version ver2.6.0a^{46 (link)}. Read counts were normalized with the reads per million per kilobase (RPKM) method. The generated gene expression matrix with RPKM scores is listed in Supplementary Data 6. DEGs were obtained using DEseq2 with default parameters^{42 (link)}. Gene expression matrix was used to perform gene set enrichment analysis (GSEA) to interpret transcriptional profiles^{47 (link),48 (link)}. Enrichment score (ES), normalized enrichment score (NES), and false discovery rate (FDR) for all variables and signatures were obtained. Gene ontology (GO) analyses were performed using an online tool DAVID (https://david.ncifcrf.gov/). The bubble plot was produced using ggplot2 (https://ggplot2.tidyverse.org/index.html) to visualize DEGs and enriched pathways.