Nebnext ultra 2 non directional rna second strand synthesis module

Total RNA was extracted using innuPREP Plant RNA Kit (Analytik Jena) from three subsamples of 100 mg from each pool to ensure sufficient total RNA yield for further processing thus allowing detection of low titre viruses. The extracted subsets were mixed and used for ribodepletion. Ribosomal RNA (rRNA) was depleted using a RiboMinus^TM Plant Kit for RNA-Seq (Invitrogen). cDNA was synthesized using ProtoScript II Reverse Transcriptase (NEB) and random octanucleotide primers (8N), followed by second strand synthesis using a NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module (NEB). The libraries were prepared from the double-stranded cDNA using Nextera XT Library Prep Kit (Illumina). The HT sequencing was performed on an Illumina MiSeq platform (301 × 2).

Samples from all 31 patients included in the Southern Medical University cohort were analyzed using RNA-seq. The formalin-fixed, paraffin-embedded (FFPE) tissue were collected before chemotherapy and radiotherapy treatment. RNA Sequencing and analysis were performed at the Genomics Laboratory of GenomicCare Biotechnology (Shanghai, China). For FFPE tissue, RNA was purified using a MagMAX FFPE DNA/RNA Ultra kit (cat# A31881, ThermoFisher), reverse-transcribed using the NEBNext RNA First Strand Synthesis Module (cat# E7525S, NEB, Ipswich, MA, USA), and NEBNext Ultra II non-directional RNA Second Strand Synthesis Module (cat# E6111S, NEB) for cDNA Synthesis. RNA-seq libraries were prepared from the cDNA using SureSelect XT HS and Low Input Library Preparation Kit for ILM (Pre PCR) (cat# G9704, Agilent, Santa Clara, CA, USA) following the manufacturer’s instructions. The libraries were sequenced on an Illumina NovaSeq-6000 Sequencing System (Illumina Inc., San Diego, CA, USA) to generate 150 × 150 paired-end reads. The expression level of each mRNA was presented as fragments per kilobase of transcript per million mapped reads (FPKM).

RNA-seq libraries were constructed with NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB E7490) and NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module (NEB E6111). RNA-seq libraries were sequenced by Illumina Nova-seq platform with pair-end reads of 150 bp. Quality control of mRNA-seq data was performed using Fatsqc (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and low-quality bases were trimmed. The adaptor sequence was removed using Cutadapt (https://cutadapt.readthedocs.io/en/stable/) to clean RNA-seq raw data. All RNA-seq data were mapped to the human reference genome (hg19) or mouse reference genome (mm10) by HISAT2 (version 2.1.0) (http://daehwankimlab.github.io/hisat2/download/). The gene expression level was calculated by Cufflinks with default parameters. Differential expressed genes (DEGs) were identified by 1.5-fold change and p value < 0.05. Gene ontology analysis and KEGG pathways analysis was performed using DAVID (https://david.ncifcrf.gov).

Double-stranded complementary DNA (cDNA) was obtained from isolated RNA by using a combination of commercial kits and primers (ProtoScript II First Strand cDNA Synthesis Kit; NEBNext Ultra II Nondirectional RNA Second Strand Synthesis Module; Random Primer 6; all New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions. Target capture of the viral cDNA was carried out with a targeted enrichment panel (SARS-CoV-2 Research Panel, Twist Bioscience, San Francisco, CA). Sequencing used NextSeq500 sequencers (Illumina, San Diego, CA, USA) to produce 2 × 150-bp reads. Raw sequencing data were converted to standard fastq format using bcl2fastq (Illumina). The adapter sequences were trimmed using the Trimmomatic 0.39 tool [17 (link)]. The DRAGEN RNA Pathogen Detection pipeline was used for alignment and variant calling [18 ]. Consensus FASTA files for four positive samples were created and deposited to the GISAID database on 8 June 2020. Phylogenetic analysis of the samples was performed as detailed previously, following the standard protocol for analysis of SARS-CoV-2 genomes as provided by Nextstrain [19 ].

Virus stocks was sequenced using a generic metagenomics sequencing workflow as described previously^{21 (link)} with some modifications. For reverse-transcribing RNA into cDNA, SuperScriptIV First-Strand cDNA Synthesis System (Invitrogen, Germany) and the NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module (New England Biolabs, Germany) were used, and library quantification was done with the QIAseq Library Quant Assay Kit (Qiagen, Germany). Libraries were sequenced using an Ion 530 chip and chemistry for 400 base pair reads on an Ion Torrent S5XL instrument (Thermo Fisher Scientific, Germany).

Extracted total RNA was sequenced using a universal metagenomics sequencing workflow (18 (link), 34 (link)). An amount of 350 ng total RNA per sample was reverse-transcribed into cDNA using the SuperScript IV first-strand cDNA synthesis system (Invitrogen, Germany) and the NEBNext Ultra II nondirectional RNA second strand synthesis module (New England Biolabs, Germany). Afterwards, cDNA was processed to generate Ion Torrent compatible barcoded sequencing libraries as described previously (2 (link), 18 (link)). Libraries were quantified with the QIAseq Library Quant assay kit (Qiagen, Germany) and subsequently sequenced on an Ion Torrent S5XL instrument using Ion 530 chips and chemistry for 400-bp reads (Thermo Fisher Scientific, Germany).

All viruses taken from archived samples were subjected to full genome sequencing essentially as described before [44 (link)]. Briefly, RNA was automatically extracted on a KingFisher Flex platform (Thermo Fisher Scientific, Waltham, MA, USA) using the RNAdvance Tissue Kit (Beckmann Coulter, Brea, CA, USA). Double stranded cDNA was generated from 350 ng total RNA using the SuperScript IV First-Strand cDNA Synthesis System (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA) and the NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module (New England Biolabs, Ipswich, MA, USA). After conversion into cDNA, fragmentation was achieved by ultrasonication on a Covaris M220 (Covaris, Brighton, UK). Subsequently, Ion Torrent-specific sequencing libraries were generated using the GeneRead L Core Kit (Qiagen, Hilden, Germany) together with IonXpress barcode adaptors (Thermo Fisher Scientific). After quantification (QIAseq Library Quant Assay Kit, Qiagen) and quality control (2100 Bioanalyzer, High sensitivity DNA Kit, Agilent Technologies, Santa Clara, CA, USA) of the libraries, sequencing was performed on an Ion Torrent S5XL instrument utilizing Ion 530 chips and reagents according to the manufacturer’s instructions.