hiscript i reverse transcriptase  (BIONOVAS Biotechnology Co Ltd)

Journal: PLOS Pathogens

Article Title: Transcriptional architecture of African swine fever virus

doi: 10.1371/journal.ppat.1014617

Figure Lengend Snippet: (A and B) Confirmation of ASFV infection in PAMs. (A) Immunofluorescence detection of the ASFV protein p72 in cells infected with ASFV. (B) Viral titers in the supernatants were measured via a hemadsorption assay and are expressed as log 10 TCID 50 /mL. Data represent the mean value ± SD. (Scale bars, 200 μm.). (C) Read counts obtained from Oxford Nanopore RNA sequencing of total RNA from ASFV-infected PAMs. (D) Distribution of read lengths at 24 and 36 hpi. (E) Correlation between host mRNA length and read alignment coverage. (F) Correlation of transcript coverage in ASFV-infected PAMs at 24 hpi and 36 hpi. Log 2 -transformed coverage was calculated using non-overlapping 50-bp genomic bins (orange points). The red line represents the linear regression fit (Pearson’s r = 0.83). (G) Nanopore long-read sequencing combined with direct RNA sequencing enables the alignment of full-length reads to the viral genome, allowing accurate mapping of TSSs and TTSs.

Article Snippet: Total RNA (1 μg) was purified and reverse-transcribed into cDNA via HiScript II Reverse Transcriptase (Vazyme, China) with a specific mix of primers following the manufacturer’s instructions.

Techniques: Infection, Immunofluorescence, RNA Sequencing, Transformation Assay, Sequencing

Journal: PLOS Pathogens

Article Title: Transcriptional architecture of African swine fever virus

doi: 10.1371/journal.ppat.1014617

Figure Lengend Snippet: (A) Nanopore long-read sequencing reveals transcriptional “read-through” at the MGF_360-1L locus. The top panel displays the RNA-seq coverage track, while the bottom panel shows individual long-read alignments. Transcription initiates at a primary transcription start site (TSS 1946) but bypasses the canonical transcription termination site (TTS 794) to terminate at a distal site (TTS 407), generating multiple RNA transcripts of varying lengths. (B) RT-PCR validation of the long transcripts. Agarose gel electrophoresis confirms the continuous transcription across the indicated genomic coordinate spans. The left panel shows PCR products from untreated total RNA, while the right panel displays PCR products from total RNA subjected to oligo(dT) reverse transcription prior to amplification. (C) Schematic representation and RT-PCR validation of the transcriptional expression of predicted novel genes. Red arrows indicate the genomic locations and transcriptional orientations of the putative novel genes. (D) Sequence logo representing the conserved promoter motif located within the 35-bp region upstream of 198 identified ASFV transcription start sites. The consensus sequence (AAAAAATTTAWWTTTTTTTW) was derived using MEME analysis (E-value = 4.8e-36). (E) Motif enrichment analysis around ASFV transcription termination sites, with a focus on 15 nucleotides upstream and 5 downstream of 259 TTSs containing continuous A/T stretches (≥7 bases).

Article Snippet: Total RNA (1 μg) was purified and reverse-transcribed into cDNA via HiScript II Reverse Transcriptase (Vazyme, China) with a specific mix of primers following the manufacturer’s instructions.

Techniques: Sequencing, RNA Sequencing, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Agarose Gel Electrophoresis, Reverse Transcription, Amplification, Expressing, Derivative Assay, Analysis

Journal: PLOS Pathogens

Article Title: Transcriptional architecture of African swine fever virus

doi: 10.1371/journal.ppat.1014617

Figure Lengend Snippet: (A) Construction of recombinant viruses harboring in-frame eGFP fusions targeted immediately upstream of the stop codons of the downstream multicistronic ORFs ( MGF_110-11L and E66L ). (B) Construction of recombinant viruses harboring in-frame eGFP fusions targeted immediately upstream of the stop codons of the potential novel viral genes ( 16848-F and 180627-R ). (C) Representative fluorescence microscopy images showing the expression of eGFP-fusion proteins in PAMs infected with the indicated recombinant ASFV strains (ASFV-ΔMGF110-11L-IneGFP, ASFV-ΔE66L-IneGFP, ASFV-Δ16848-F-IneGFP, and ASFV-Δ180627-R-IneGFP). Scale bars: 200 μm. (D) RT-PCR validation of the target gene-eGFP junctions reveals that the transcripts of multicistronic downstream ORFs and potential novel viral genes in the recombinant viruses contain the eGFP sequence, generating transcripts containing the target gene–eGFP junction. Total RNA was extracted from PAMs infected with the respective recombinant viruses. Left: PCR amplification using non-reverse-transcribed RNA as template (negative control to exclude viral genomic DNA contamination). Right: PCR amplification of cDNA reverse-transcribed with oligo(dT) primers, using specific primers targeting the eGFP sequence. M: DNA marker.

Article Snippet: Total RNA (1 μg) was purified and reverse-transcribed into cDNA via HiScript II Reverse Transcriptase (Vazyme, China) with a specific mix of primers following the manufacturer’s instructions.

Techniques: Recombinant, Fluorescence, Microscopy, Expressing, Infection, Reverse Transcription Polymerase Chain Reaction, Biomarker Discovery, Sequencing, Amplification, Reverse Transcription, Negative Control, Marker