Ratios of R203/G204: 203K/204R RNA were determined via RT-PCR with quantification of Sanger peak heights. Briefly, R203/G204 and 203K/204R viruses were mixed at PFU ratios of 1:1, 3:1 and 9:1 based on their PFU titers. To quantify R203/G204: 203K/204R ratios, a 596 bp RT-PCR product (Primers: SARS-CoV-2 28354F, 5¢-CCAGAATGGAGAACGCAGTG-3¢; SARS-CoV-2 28949R, 5¢-TGTCAAGCAGCAGCAAAGC-3¢) was amplified from the extracted RNA using a SuperScript III One-Step RT–PCR kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The PCR product was purified by a GeneJET PCR Purification kit (Thermo Fisher Scientific) and submitted to Sanger sequencing (BGI, Shanghai, China) (Primer for Sanger sequencing: 5¢-CCAGAATGGAGAACGCAGTG-3¢). The sequence electropherograms were further scored by QSV analyzer to quantify the proportion of R203/G204 and 203K/204R viruses. The correlation between input PFU ratios and output RT–PCR amplicon ratios and verification of the actual ratios of R203/G204: 203K/204R achieved upon viral mixing are shown in Figures S6 A and S6B.
Sanger sequencing
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Sanger sequencing is a method for determining the nucleotide sequence of DNA. It involves the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.
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Lab products found in correlation
Topo ta cloning kit, by Thermo Fisher Scientific (4 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Genejet plasmid miniprep, by Thermo Fisher Scientific (2 mentions)
Q5 dna polymerase, by New England Biolabs (2 mentions)
Superscript 3 reverse transcriptase kit, by Thermo Fisher Scientific (2 mentions)
Pcr blunt 2 topo vector, by Thermo Fisher Scientific (2 mentions)
Phusion high fidelity pcr master mix, by New England Biolabs (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (2 mentions)
Stbl3 cells, by Thermo Fisher Scientific (1 mentions)
Single stranded oligos, by Merck Group (1 mentions)
Gibson cloning kit, by New England Biolabs (1 mentions)
Neb hifi assembly, by New England Biolabs (1 mentions)
Snapgene software, by Dotmatics (1 mentions)
Hifi assembly, by New England Biolabs (1 mentions)
Superscript 3 first strand synthesis supermix for qrt pcr, by Thermo Fisher Scientific (1 mentions)
Faststart universal sybr green master mix, by Roche (1 mentions)
69 protocols using sanger sequencing
1
Quantifying SARS-CoV-2 Variant Ratios via RT-PCR
2
Codon-Optimized Base Editing Constructs
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Human codon-optimized base editing constructs were a kind gift from David Liu; pCMV_ABEmax_P2A_GFP (plasmid #112101; Addgene), pCMV_AncBE4max_P2A_GFP (plasmid#112100; Addgene). pCMV_SpCas9-NG_ABEmax_P2A_GFP, pCMV_SpCas9-NG_AncBE4max_P2A_GFP and pCMV_SaKKH_AncBE4max_P2A_GFP were constructed by PCR amplification (Q5, NEB) amplifying everything except for SpCas9 using pCMV_ABEmax_P2A_GFP and pCMV_AncBE4max_P2A_GFP. Coding sequences for SpCas9-NG and SaKKH were PCR amplified using the following plasmids NG-ABEmax (plasmid #124163; Addgene) and SaKKH-ABEmax (Plasmid #119815; Addgene) that were a kind gift from David Liu. Coding sequences and plasmid backbones were combined using the NEBbuilder HiFi DNA assembly mastermix (NEB) and subsequently transformed using OneShot Mach1t1 (Thermo Fisher Scientific) cells and plasmid identity was checked by Sanger sequencing (Macrogen). The empty sgRNA plasmid backbone for SpCas9 and its derivatives was a kind gift from Keith Joung (BPK1520, Addgene plasmid #65777). Spacer sequences targeting all genes in this study were cloned in the sgRNA plasmid backbone using inverse PCR (Q5 NEB) and subsequently transformed using OneShot Mach1t1 (Thermo Fisher Scientific) cells and plasmid identity was checked by Sanger sequencing (Macrogen). Primer sequences for sgRNA generation can be found in Supplementary Table 3 .
3
Rabies Virus Detection and Sequencing
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Selected brain samples were sent to AHVLA for independent confirmation of results (Table 1 ). Extracted nucleic acids (Trizol, Invitrogen) were tested by a differential real-time Taqman RT-PCR assay as previously described using the primers JW12, N165-145 and a RABV specific probe [28] (link). A 606 bp region of the N-gene of positive samples was amplified by hemi-nested RT-PCR [29] and the full nucleoprotein of a subset of isolates (n = 23) was then derived using Sanger sequencing (ABI) with overlapping primers designed from a Grenadian strain (primer sequences available on request). At least one forward and one reverse primer were used to derive consensus sequences, which were then aligned using CLUSTALX.
4
Cloning CRISPR Lentiviral Guides
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LentiGuide-Puro was digested with BsmBI and purified by gel extraction (Macherey-Nagel). Single stranded oligos (Sigma) containing the guide sequence and 25nt overlap with digested LentiGuide-Puro on each side (Supplementary Table 1 ) were cloned with the Gibson cloning kit (NEB). Chemo-competent Stbl3 cells (Thermofisher) were transformed with the Gibson products by heat shock. Upon minipreps (Macherey-Nagel), the plasmids were verified by Sanger sequencing (ABI).
5
Generating pFlyORF-TaDa and pTaDaG2-MRG15 vectors
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The pFlyORF-TaDa vector was created by cutting pTaDaG2 (Delandre et al. 2020 ) with BglII/NotI (NEB) and inserting a synthetic gBlock (IDT) containing an FRT5 site followed by an in-frame stop codon via NEB Hifi Assembly (NEB) to create pTaDaG2-FRT5. pTaDaG2-FRT5 was cut with AfeI/BstBI and a synthetic gBlock (IDT) containing 3xP3-DsRed2-small_t_intron-polyA via NEB Hifi Assembly to generate pTaDaG2-FRT5-3xP3-dsRed2. Finally, mini-white (including the 240 bp upstream and 630 bp downstream white regulatory regions) was amplified via PCR from pTaDaG2 and inserted into pTaDaG2-FRT5-3xP3-dsRed cut with XhoI and XbaI via NEB Hifi Assembly to generate pFlyORF-TaDa.
pTaDaG2-MRG15 was generated by inserting a synthetic gBlock (IDT) containing the sequence of MRG15-RA into the pTaDaG2 vector cut with XbaI/XhoI. All plasmids were sequence-verified via Sanger sequencing (ABI). Plasmid maps were generated using SnapGene software (Insightful Science).
pTaDaG2-MRG15 was generated by inserting a synthetic gBlock (IDT) containing the sequence of MRG15-RA into the pTaDaG2 vector cut with XbaI/XhoI. All plasmids were sequence-verified via Sanger sequencing (ABI). Plasmid maps were generated using SnapGene software (Insightful Science).
6
qPCR Characterization of hESC Lines
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Total RNA extracted from three hESC lines (H9, HS401 and HS980) in three biological replicates of each was converted to cDNA using SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen, 11752) according to the manufacturer's instructions. qPCR assay was performed using 22 ng cDNA from each cell line sample and 10 ng human 8-cell library that was used for cloning of the genes. qPCR was carried out using an ABI PRISM 7500 Fast Real-Time PCR System with FastStart Universal SYBR Green Master Mix (Roche) according to the manufacturer's instructions. The primer sequences are given in Table S4 . To confirm the qPCR amplicon, it was cloned into pCRII-dual promoter TOPO vector using the TOPO TA cloning kit (Invitrogen), and the sequence was verified by Sanger sequencing (Eurofins Genomics).
7
STC2 Overexpression and Knockdown Lentivirus Construction
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The full-length coding sequence of STC2 was cloned into pLV-IRES-eGFP vector, and the shRNA of STC2 was inserted into pLKO.1 vector. The shRNA sequence of STC2 was 5′- AGG GCA AGT CAT TCA TCA AAG C -3′. The constructs were confirmed by Sanger sequencing in Invitrogen. And the packaging and infection of overexpression and knock-down vectors were briefly described as follows: the overexpressing pLV-IRES-eGFP-STC2 or knocking down plasmids pLKO.1-STC2 were, respectively, cotransfected with pCMV-VSV-G and pCMV-deltaR8.91 plasmids into HEK293T cells with lipofectamine 2000 and cultured for 48 h. The supernatant was collected and filtrated through 0.45 μm filter membrane to remove the cells and fragments. Then the collected solution was subjected to lentivirus concentration solution kit according to the manufacturer's instruction. The enriched lentivirus solution was stored in ultralow temperature freezer.
8
Temporal Transcriptional Profiling of Tephritid Fly
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Adult males were sampled daily from day 1 to 30 after emergence (DAE). Total RNA was isolated with TRIzol reagent (Invitrogen) following the manufacturer’s instructions. Total RNA was incubated with 10 U DNase I (Thermo Scientific, USA) at 37 °C for 30 min for mRNA purification. First strand cDNA was produced from 5 μg RNA using Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). Target gene sequences (lola, topi, per, aly, rac, rho, upd, magu) were obtained by retrieving previously constructed B. dorsalis transcriptome data (Y.-C. D., Z.-J. W., C.-Y. N., unpublished data), and their gene specific primers were designed using Primer Premier 5.0 (Premier, Canada) (Table S1). PCR amplicons were purified using AxyPrep DNA Gel Extraction Kit (AxyPrep, USA). The purified products were ligated to cloning vector by using pMD™ 18-T Vector Cloning Kit (TaKaRa, China). The plasmid recombinants (pMD-18T-lola, -topi, -per, -aly, -rac, -rho, -upd, -magu) were amplified by PCR and verified by Sanger sequencing (Invitrogen, Shanghai, China).
9
Lentiviral Transduction of SOCS3 with Tags
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We created lentiviral constructs to express SOCS3 with C-terminal Myc and DDK tags by subcloning the human SOCS3 coding sequence and tGFP in a single mRNA using a P2A linker following Origene’s TrueORF cloning instructions. Briefly, SOCS3 cDNA was cut with EcoRI and XhoI and subcloned into pLenti-C-Myc-DDK-P2A-tGFP lentiviral gene expression vector. A lysine 6 mutation (K6Q-SOCS3) was obtained by PCR mutagenesis using PfuUltra II Fusion High-fidelity DNA Polymerase. All constructs were confirmed by Sanger sequencing (Genewiz).
Lentiviral particles were grown in HEK293FT cells (Invitrogen, Thermo Fisher Scientific, R700-07) by cotransfecting cells with the SOCS3-expressing lentiviral constructs or pLenti-C-Myc-DDK-P2A-tGFP (empty vector control) and pCMV-dR8.2 dvpr and pCMV-VSVG packaging plasmids (98 (link)). After 48 hours, medium was concentrated using a 30 kDa cutoff filter, aliquoted, and stored at –80°C until use. Viral titer was determined by the proportion of infected cells with green fluorescence 72 hours postinfection.
Lentiviral particles were grown in HEK293FT cells (Invitrogen, Thermo Fisher Scientific, R700-07) by cotransfecting cells with the SOCS3-expressing lentiviral constructs or pLenti-C-Myc-DDK-P2A-tGFP (empty vector control) and pCMV-dR8.2 dvpr and pCMV-VSVG packaging plasmids (98 (link)). After 48 hours, medium was concentrated using a 30 kDa cutoff filter, aliquoted, and stored at –80°C until use. Viral titer was determined by the proportion of infected cells with green fluorescence 72 hours postinfection.
10
Genetic Variants in Hypertriglyceridemia
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Blood samples obtained from the proband were sent to the Nanfang Hospital Precision Medicine Center for Whole-exome sequencing (WES) of genomic DNA. Illumina HiSeq platform was used for WES. The criteria established and revised by the American College of Medical Genetics and Genomics (Richards et al., 2015 (link)) was used to classified the variants. Two hypertriglyceridemia associated genes mutations, within exon 10 of the LMF1 gene and exon 5 of the LPL gene identified by WES, were verified using Sanger sequencing. Standard phenol/chloroform extraction was performed to extract genomic DNA from the peripheral blood acquired from the proband and his family members (Ⅰ1, Ⅰ2, Ⅱ4, Ⅱ2, Ⅲ4, and Ⅲ3). The PCR primers that were designed using Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast ) were as follows: LMF1: Exon-10 forward primer: 5′-CCGTCTCAGCCACCAGAAAA-3′, Exon-10 reverse primer: 5′-CACGGCTGGTTTGGTTTGAG-3'; and LPL: Exon-5 forward primer: 5′-CCAGCCATCCTGAGTGGAAA-3′, Exon-5 reverse primer: 5′-GGCTCTAAGGTGGTCATGCT-3'.The PCR products were then analyzed by agarose gel electrophoresis and submitted to Invitrogen (Shanghai, China) for Sanger sequencing.
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