Viral RNA was extracted from virus supernatants using the NucleoSpin RNA Virus kit (Macherey-Nagel), reverse transcribed with AccuScript (Agilent Technologies), and used for PCR amplification. The viral genome was amplified using eight different, overlapping, primer pairs (sequence available upon request). PCRs were column purified and used for Sanger sequencing. Chromatograms were analyzed with the Staden package (http://staden.sourceforge.net ).
Accuscript
Manufactured by Agilent Technologies
The AccuScript is a high-performance molecular biology enzyme that offers accurate and efficient reverse transcription for RNA analysis. It is designed to deliver consistent cDNA synthesis with enhanced sensitivity and reliability.
Automatically generated - may contain errors
Lab products found in correlation
Nucleospin rna virus kit, by Macherey-Nagel (2 mentions)
Hiseq 2500, by Illumina (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Miseq sequencing, by Illumina (1 mentions)
Qiaquick gel extraction kit, by Illumina (1 mentions)
Facsaria 2, by BD (1 mentions)
Genejet rna purification kit, by Thermo Fisher Scientific (1 mentions)
Anti hla a2 pe clone bb7, by BioLegend (1 mentions)
Generuler 1 kb plus dna ladder, by Thermo Fisher Scientific (1 mentions)
6 protocols using accuscript
1
Viral RNA Extraction and Genome Sequencing
2
Amplification and deep sequencing of CXCR4/CCR5
3
Intracellular Viral RNA Quantification
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Total intracellular viral RNA was extracted from infected cells using the Qiagen RNeasy kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions. RT-PCR was carried out using AccuScript (Agilent Technologies), with specific oligonucleotide primers (S1 Table ) The amplicons covered the following genomic regions: A1, spanning genomic residues 7626 to 7962; A2, residues 7941 to 8257; and A3, residues 8229 to 8653. Negative controls without template RNA were included in parallel to ascertain the absence of cross-contamination by template nucleic acids. PCR products were purified (QIAquick Gel Extraction kit), quantified (Pico Green assay), and analyzed for quality (Bioanalyzer) prior to Illumina MiSeq sequencing.
4
Tapasin-KO Expi293F Cell Screening
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Tapasin-KO Expi293F cells expressing the tapasin or TAPBPR-CT libraries were stained with diluted 1:200 anti-HLA-A2 PE (clone BB7.2, BioLegend Cat# 343302) in PBS-BSA for 30 min at 4 °C and washed twice with PBS-BSA. After gating for the main population by forward-side scatter and excluding dead cells based on DAPI uptake, the top 0.5% of cells for PE fluorescence were collected using a BD FACSAria II. RNA was extracted from sorted cells (GeneJet RNA purification kit, ThermoFisher), first-strand cDNA was synthesized using Accuscript (Agilent) primed with the EBV reverse sequencing primer, which anneals to the 3′ UTR of pCEP4-encoded transcripts. DNA fragments covering mutated regions were PCR amplified in two rounds to append sequences complementary to Illumina sequencing primers (Supplementary Table 2 ) and add experiment-specific barcodes and Illumina adaptamers. Products were sequenced on a NovaSeq 6000 and analyzed with Enrich49 (link). Scripts for analysis are included with the GEO submission.
5
RT-PCR Amplification and Sequencing Protocol
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RT-PCR amplification was carried out using AccuScript (Agilent) following the manufacturer's instructions. Amplification products were analyzed by agarose gel electrophoresis, using GeneRuler 1 kb Plus DNA Ladder (Thermo Scientific) as molar mass standard. Amplification controls in the absence of RNA were run in parallel to ascertain the absence of contamination by undesired templates. Amplified DNA was sequenced using the 23 ABI 3730 XLS sequencer (Macrogen, Inc.). All the oligonucleotide primers used in this study are listed in Supplementary Table S1 .
6
SARS-CoV-2 Genome Sequencing Protocol
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Viral RNA was extracted from infected supernatants using the NucleoSpin RNA Virus kit (Macherey-Nagel), reverse-transcribed with AccuScript (Agilent Technologies), and used for PCR amplification. The genome was amplified using four overlapping PCR fragments (primer sequences available upon request), which were column-purified and used for Sanger sequencing. Chromatograms were analyzed using the Staden software (http://staden.sourceforge.net ).
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