Nebnext ultra 2 rna library prep kit

To test the effect of the rsmA and rsmE genes on the transcriptome in P. fluorescens 2P24, cells were cultured to early stationary phase (OD₆₀₀ = 1.0) in LB medium. Total RNA was conducted using the RNeasy minikit (Qiagen, MD, U.S.A.). The Ambion Turbo DNA-free kit was applied to remove contaminant DNA. After removal of rRNA by using the Ribo-Zero rRNA removal kit (Illumina, CA, U.S.A.), mRNA was used to generate the cDNA library according to NEBNext UltraTM II RNA Library Prep Kit, which was then sequenced using an Illumina HiSeq 2500 platform. High-quality reads were aligned to the P. fluorescens 2P24 genome (GenBank accession no. CP025542). From the resulting alignments, SAMtools version 1.6 [40 (link)] was applied to sort the bam file. The differentially expressed genes were identified by performing Cuffdiff version 2.2.1 [41 (link)] with a p value smaller than 1e-5. Each sample in the RNA-seq was repeated three times.

RNA was extracted from KYSE150 and KYSE180 cells using TRIzol (Beyotime, China). The extracted RNA samples were first examined for concentration and purity to exclude degradation or contamination using a Qubit and Nanodrop spectrophotometer. For library construction, nonstranded cDNA libraries were constructed using poly(A) mRNA enrichment and the NEBNext UltraTM II RNA Library Prep Kit for Illumina following the manufacturer’s instructions. RNA and library qualities were confirmed using fragment analysis (Agilent 2100 Bioanalyzer). FastQC software (version 0.11.7) was used for quality control of the raw data, and Illumina was used to evaluate the sequencing error rate and base quality. For gene differential expression analysis, EBSeq was used to obtain the differentially expressed gene sets between the two samples, and fold change ≥ 2 or ≤ 1/2 and FDR < 0.01 were used as the screening standards. To investigate differences in the biological processes associated with these DEGs, Gene Ontology (GO) enrichment was performed (Q ≤ 0.05 was considered to indicate significant enrichment). Next, the enriched annotated genes were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) functional analysis.

Transcriptional analysis of the patient tumor biopsies has been described elsewhere (44 (link)). Briefly, patient tumor biopsies were either fresh-frozen and stored in liquid nitrogen or immediately treated with RNAlater (Thermo Fisher Scientific) and stored at –80°C. Each RNAlater-treated sample was homogenized with the QIAGEN TissueRuptor II, and RNA was isolated with the AllPrep DNA/RNA Mini Kit (QIAGEN). The NEBNext Ultra II RNA Library Prep Kit for Illumina was used to prepare sequencing libraries. Paired-end sequencing (PE150) was performed on the NovaSeq 6000 system (Illumina). Sequences were mapped and quantified to the decoy-aware, concatenated transcriptome of GRCh38.p13 (Ensembl, version 102) and MCPyV (R17b) using Salmon (78 (link)). Gene-level counts were generated via TxImport (79 (link)), and normalized counts were generated via DESeq2 (80 (link)). Samples were considered virus positive if the number of normalized MCPyV LT counts was greater than 100 and the number of normalized ST counts was greater than 10.

Suit-2 cells were treated with iBet-762 (2 µM), OTX-015 (1 µM), Neo-2734 (0.5 µM), or DMSO (ctrl) for 12 h alone or infected with Ad5wtGFP for 2 h alone, or inhibitors were added to the infected cells after 2 h and cultured for another 12 h. Ad5wtGFP-infected cells were harvested and GFP+ cells were collected by FACS sorting. Total RNA was extracted using the Trizol reagent (Invitrogen), quantified, and RNA integrity determined by Tapestation (Agilent, Santa Clara, CA, USA). Samples with an RNA Integrity Number (RIN) >8.0 were used for the RNA sequencing library preparation and mRNA was enriched by Poly(A) mRNA magnetic beads (New England BioLabs, Ipswich, MA, USA). The NEBnext Ultra II RNA Library Prep Kit for Illumina (E7770, San Diego, CA, USA) was used to generate RNA sequencing libraries. Libraries were sequenced for 37 bp paired end by NextSeq 500 high output Run, with an average of 30 million reads per sample.

Total RNA was extracted from each cell population using the NucleoSpin RNA Mini kit (MACHEREY-NAGEL). Then, secondary RNA purification was performed using the NEB Next Poly A m-RNA magnetic bead selection kit (E7490), and each cDNA library was prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina (E7770). Sequencing was performed using the Illumina Nextseq 500 platform with paired-end 75-bp reads. Sequenced reads were aligned to the mm10 reference genome using Bowtie2 (Langmead and Salzberg, 2012 (link)) with default parameters. Rsubread R package (Liao et al., 2019 (link)) was used to count the number of RNA-Seq reads. Screening of the differentially expressed genes, clustering analysis, and PCA analysis were performed using DESeq2 (Love et al., 2014 (link)) on the iDEP9.1 platform (Ge et al., 2018 (link)). Functional enrichment analysis was performed on Metascape (Mizukami et al., 2016 (link); Zhou et al., 2019 (link)).
The RNA-Seq datasets of FSCs or EpiSCs cultured in N2B27-based medium in the previous study (Kinoshita et al., 2021 (link)) were used for PCA analysis.