Nebnext ultra rna library preparation kit for

Whole genome RNA was extracted using the TRIZOL method, RNA concentration was determined using NanoDrop 2000 (Thermo), and RNA integrity was detected using an Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). The sequencing library was built using the NEBNext Ultra RNA Library Preparation Kit for Illumina (NEB, USA) and library quality was assessed on an Agilent Bioanalyzer 2100 system. Clustering was performed using the TruSeq PE Cluster Kit v4-cBot-HS (Illumia), after which the library preparations were sequenced on an Illumina Hiseq Xten platform and the readings at the paired ends were generated.

Cells were sorted into TRIzol-LS (Invitrogen) and total RNA was purified using RNeasy micro kit (Qiagen) and analyzed for integrity using RNA 6000 pico kit for 2100 Bioanalyzer (Agilent). mRNA was purified from total RNA using NEBNext Poly(A) mRNA magnetic isolation module (NEB). Poly(A)-selected mRNA was fragmented to an average size of 300 nt, reverse-transcribed, and converted to a paired-end sequencing library using NEBNext Ultra RNA library preparation kit for Illumina (NEB) according to the vendor's instructions. Libraries were sequenced on an Illumina HiSeq 2500 sequencer in paired-end mode with the read length of 75 nt.

Total RNA was extracted from leaf samples preserved in liquid nitrogen using an RNAprep Pure Plant Kit (Tiangen DP441), and genomic DNA contamination was removed using DNase I (Tiangen). RNA degradation and contamination was monitored on 1% agarose gels. RNA purity was checked using the NanoPhotometer^® spectrophotometer (IMPLEN, CA, USA). RNA concentration was measured using Qubit^® RNA Assay Kit in Qubit^®3.0 Flurometer (Life Technologies, CA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). The isolated 1 µg RNA was used for cDNA library construction using the NEBNext Ultra RNA Library Preparation Kit for Illumina (New England Biolabs, Ipswich, MA, USA), with fragment lengths of approximately 150 bp. The cDNA library was paired-end sequenced using an Illumina NovaSeq 6000 platform.

Total RNA was extracted from BMDM cultures infected with ASFV CN/GS 2018 or ASFV-GS-Δ18R/NL/UK using TRIzol reagent (Invitrogen). Thereafter, quantification and qualification were performed. Three micrograms of RNA per sample was used for library preparation using the NEBNext Ultra RNA library preparation kit for Illumina (New England Biolabs [NEB]), according to the manufacturer’s recommendations. The libraries were sequenced on an Illumina HiSeq platform, and 150-bp paired-end reads were generated. Differential expression analysis was performed using the DESeq2 R package (version 1.16.1). The resulting P values were adjusted using Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with an adjusted P value of <0.05, found by DESeq2, were determined to be differentially expressed. Gene Ontology (GO) enrichment analysis of DEGs was performed using the topGO R package, where gene length bias was corrected. GO terms with P values of <0.05 were considered significantly enriched by DEGs. The ClusterProfiler R package was used to test the statistical enrichment of DEGs in the KEGG pathways.

Sequencing libraries were prepared using the NEBNext^® Ultra™ RNA Library Preparation Kit for Illumina^® (NEB, United States) following the manufacturer’s recommendations, and index codes were added to the attribute sequences of each sample. After column purification, the quality of the obtained libraries was assessed on an Agilent Bioanalyzer 2100 system. Libraries are sequenced on the Illumina Hiseq 2000 platform. These reads are available from the NCBI Sequence Read Archive, BioProject Accession Number PRJNA892217 and PRJNA612421.

Total RNA was isolated from the leaf tissues of control and waterlogged plants of both genotypes by using the RNeasy Plant Mini Kit (Qiagen, Germany). For each treatment, RNA was isolated from the leaf samples of three plants that were considered as one replicate. Three such independent biological replicates were used in the present study. Total RNA from each replicate was quantified and qualified using NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA, USA) and through 1% agarose gel analysis. Further, the RIN value of the isolated RNA was determined using Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). An equal amount of high-quality RNA from each replicate of the control and stressed samples of each genotype was used for library preparation. In total, 12 next-generation sequencing libraries were prepared using the NEBNext^®Ultra™ RNA Library Preparation Kit for Illumina^® (NEB, Ipswich, MA, USA) as per the manufacturer’s protocol. The libraries were then sequenced in the paired-end mode with 150 × 2 read length by using the Illumina Hi-Seq 2500 platform.