Nebnext ultra 2 non directional rna second strand synthesis module kit

Total RNA (totRNA) was extracted from N. benthamiana infected leaf material using innuPREP RNA Mini Kit (Analytik Jena AG, Jena, Germany) following the manufacturer’s protocol. Ribosomal RNA (rRNA) was depleted using RiboMinus Plant kit (Invitrogen, Carlsbad, CA, USA) according the manufacturer’s protocol. Random cDNA was synthesized using ProtoScript II Reverse Transcriptase (New England Biolabs, Beverly, MA, USA) and 8 N random primers. The second-strand was synthesized with NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module kit (New England Biolabs (NEB), Beverly, MA, USA). A library was prepared using Nextera XT Library kit (Illumina) and subsequently run on a MiSeq v3 platform as pair-end reads (2 × 301).

Total RNA was extracted from cell-culture supernatants using the QIAamp^® Viral RNA mini kit (Qiagen, Hilden, Germany) and used for the synthesis of the first cDNA strand using SuperScript III reverse transcriptase and random primer hexamers (Invitrogen, Life Technologies, Carlsbad, CA, USA). The second cDNA strand was obtained using the NEBNext^® Ultra™ II Non-Directional RNA Second Strand Synthesis Module kit (New England BioLabs, Hitchin, UK). Library preparation was then completed with the NEBNext^® Ultra™ II RNA Library Prep Kit for Illumina (New England BioLabs). After quality validation on the 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA), the library was sequenced to obtain 2 × 150 bp paired-end reads on an Illumina NovaSeq 6000 sequencer (Integragen, Ivry, France).

Two groups of 786-O cell-derived exosmes containing oe-AP000439.2 or oe-NC were subjected to incubate with macrophages, and then these pretreated macrophages were performed transcriptome sequencing with triple biological repetitions. The extraction of total RNA was prepared with NEBNext Poly(A) mRNA kits (E7490, NEB) followed by treated with NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module kit (E6111, NEB) to synthesis the second strand of cDNA. Next, the cDNA libraries were constructed using Vazyme kit (ND604, Vazyme). Libraries were sequenced by Illumina Hiseq X Ten platform. Raw data were quality controlled using Fast-QC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Clean data were mapped to the human genome (hg38) by TopHat (version 2.1.1, http://ccb.jhu.edu/software/tophat/index.shtml). The gene expression level was calculated by Cufflinks, and when the gene expression satisfied the criteria for |log2 fold change|> 1 and false discovery rates < 0.05, it was considered a differentially expressed gene (DEG). The pathway analysis of DEG was performed by DAVID (https://david.ncifcrf.gov/).