The 3′ untranslated region (UTR) of the PPARG gene, containing the bta-miR-128 binding site, was amplified by PCR using bovine genomic DNA as the template. The amplicon was purified then ligated into the XhoI–NotI site of the psiCHECKTM-2 vector. The resulting plasmid was used to transform E. coli DH5α. Using “white-blue colony selection,” white colonies were cloned then amplified. The final recombinant plasmid was named PPARG-3′UTR-WT. The seed region of the bta-miR-128 binding site was mutated (Tsingke Company) and PPARG-3′UTR-Mut was constructed. The psiCHECKTM-2 reporter plasmid was a gift from Dr. Guirong Sun. Similarly, luciferase vectors of solute carrier family 16 member 1 were constructed (SLC16A1-3′UTR-WT and -Mut); SLC16A1-3′UTR-Mut was constructed using primer mutation. All plasmids were extracted using an EndoFree Mini Plasmid Kit II (TIANGEN, Beijing, China) and were sequenced by Biosunya Biotechnology Co. Ltd. (Shanghai, China). Primers are listed in Supplementary Table S1 .
Endofree mini plasmid kit 2
Manufactured by Tiangen Biotech
Sourced in China
The EndoFree Mini Plasmid Kit II is a laboratory equipment used for the purification of plasmid DNA from bacterial cultures. It is designed to efficiently remove endotoxins, providing high-quality plasmid DNA suitable for various downstream applications.
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Lab products found in correlation
Universal dna purification kit, by Tiangen Biotech (2 mentions)
X tremegene hp dna transfection reagent, by Roche (2 mentions)
Pmd18 t vector, by Takara Bio (2 mentions)
Hiscript 2 q rt supermix for qpcr, by Vazyme (2 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Pspcas9 bb 2a gfp px458 plasmid, by Addgene (1 mentions)
Gel extraction kit, by Omega Bio-Tek (1 mentions)
Dna ligation kit, by Takara Bio (1 mentions)
Hieff trans liposomal transfection reagent, by Yeasen (1 mentions)
Psicheck 2 vector, by Promega (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Hepg2 cell, by American Type Culture Collection (1 mentions)
Hyclone, by GE Healthcare (1 mentions)
Grace s medium, by Thermo Fisher Scientific (1 mentions)
Taq pcr starmix, by GenStar (1 mentions)
Sf 900 3 sfm, by Thermo Fisher Scientific (1 mentions)
Yeastmaker yeast transformation system 2, by Takara Bio (1 mentions)
Trans t1 phage resistant chemically competent cells, by Transgene (1 mentions)
T4 dna ligase, by Takara Bio (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
20 protocols using endofree mini plasmid kit 2
1
Cloning and Mutagenesis of PPARG and SLC16A1 3'UTRs
2
CRISPR/Cas9 Genome Editing: Targeting DHBV
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sgRNAs were designed using the CRISPR/Cas system (Cas9/gRNA) Off-Targeter (CasOT) tool (http://casot.cbi.pku.edu.cn/ ; Peking University, Beijing, China) to minimize potential off-target effects. The sequences of six sgRNAs targeting DHBV genome (GenBank: K01834.1) and one nonsense sgRNA (sgNS) are presented in Table I .
The PSpCas9(BB)-2A-GFP (PX458) plasmid was obtained from Addgene, Inc. (Cambridge, MA, USA). The plasmid was extracted using the EndoFree Mini Plasmid Kit II (Tiangen Biotech Co., Ltd., Beijing, China). PX458 was digested with BbsI (New England Biolabs, Inc., Ipswich, MA, USA), and then the linearized vector was purified using the Gel Extraction kit (Omega Bio-tek, Inc., Norcross, GA, USA) according to the manufacturer's instructions. Each pair of sgRNAs was annealed to double strands, which was ligated to the linearized vector with T4 DNA ligase (New England Biolabs, Inc.). The co-expression plasmid sgRNA/Cas9 was identified through sequencing (Fig. 1 ).
The PSpCas9(BB)-2A-GFP (PX458) plasmid was obtained from Addgene, Inc. (Cambridge, MA, USA). The plasmid was extracted using the EndoFree Mini Plasmid Kit II (Tiangen Biotech Co., Ltd., Beijing, China). PX458 was digested with BbsI (New England Biolabs, Inc., Ipswich, MA, USA), and then the linearized vector was purified using the Gel Extraction kit (Omega Bio-tek, Inc., Norcross, GA, USA) according to the manufacturer's instructions. Each pair of sgRNAs was annealed to double strands, which was ligated to the linearized vector with T4 DNA ligase (New England Biolabs, Inc.). The co-expression plasmid sgRNA/Cas9 was identified through sequencing (
3
Generation and Validation of APOBEC3C Overexpressing Cell Lines
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Wild-type APOBEC3C consensus coding sequence (CCDS) (APOBEC3C-pENTER vector, WZ Biosciences) was amplified by PCR (forward primer: 5’-CCGTCAGATCCGCTAGTAATAC-3’, reverse primer: 5’-CCGGAATTCTGTGGTATGGCTGATTATGATC-3’). Then, BamHI and EcoRI were employed to generate sticky ends. The pCDH-CMV-puro plasmid was digested with the same enzymes, and restriction enzyme-digested products were purified by electrophoresis and Universal DNA Purification Kits (Cat#DP214-03, Tiangen, China). The CCDS fragment and pCDH plasmid were ligated using DNA Ligation Kits (Cat# 6021, Takara, Japan) and then transformed into DH5α cells for further selection and amplification.
EndoFree Mini Plasmid Kit II (Cat# DP118-02, Tiangen) was used to extract pCDH-APOBEC3C (pCDH-A3C). Then, 7.5 µg pCDH-A3C or pCDH backbone plus 5.625 µg psPAX2 and 1.875 µg pMD2.G were incubated with 30 µL Hieff Trans Liposomal Transfection Reagent (Cat# 40802ES03, Yeason, China) in 1 mL DMEM. The mixture was added to 293T cells to generate lentiviruses, which were used to transfect pancreatic cell lines. Puromycin was added to culture mediums (4 µg/mL) for selection. Four months after transfection, total DNA was extracted from A3C-overexpressing cell lines and subjected to Sanger sequencing to verify that A3C sequence in the plasmid did not undergo genetic modifications.
EndoFree Mini Plasmid Kit II (Cat# DP118-02, Tiangen) was used to extract pCDH-APOBEC3C (pCDH-A3C). Then, 7.5 µg pCDH-A3C or pCDH backbone plus 5.625 µg psPAX2 and 1.875 µg pMD2.G were incubated with 30 µL Hieff Trans Liposomal Transfection Reagent (Cat# 40802ES03, Yeason, China) in 1 mL DMEM. The mixture was added to 293T cells to generate lentiviruses, which were used to transfect pancreatic cell lines. Puromycin was added to culture mediums (4 µg/mL) for selection. Four months after transfection, total DNA was extracted from A3C-overexpressing cell lines and subjected to Sanger sequencing to verify that A3C sequence in the plasmid did not undergo genetic modifications.
4
Validating miRNA-Target Gene Interactions
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The target sequences were predicted using NCBI database, and the primers of these target genes MEIS2 (Meis Homeobox 2), MAPK7 (Mitogen-Activated Protein Kinase 7), UCP2 (Uncoupling Protein 2), SNX1 (Sorting Nexin 1), CPS1 (Carbamoyl-Phosphate Synthase 1), HACD3 (3-Hydroxyacyl-CoA Dehydratase 3), WNT4 (Wnt Family Member 4), CAMK2B (Calcium/Calmodulin Dependent Protein Kinase II Beta), TCF7 (Transcription Factor 7) were designed using Primer 5. The primer information of mmu-miR-24-3p and bta-miR-24-3p were listed in Tables S6 and S7 . The genomic DNA mRNA 3′-UTR sequence of these target genes was cloned from genomic DNA using polymerase chain reaction (PCR) amplification. When we obtained the correct DNA products, we inserted these target genes’ genomic DNA into the psiCHECK2 vector (Promega, Madison, WI, USA). The recombinant vectors were extracted using an Endofree Mini Plasmid Kit II (Tiangen, Beijing, China) extraction kit, followed by confirmation with enzyme digestion sequencing. After sequencing verification, the successfully constructed vectors were used for cell transfection. The transfection procedure was performed following the manufacturer’s instructions for the transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA).
5
HepG2 Cell Transfection Protocol
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HepG2 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s Modified Eagle Medium (HyClone, GE, Beijing, China) with 10% fetal bovine serum (HyClone, GE, Beijing, China). Transfection reactions were performed when 1 × 106 cells reached 80% confluency in 6-well plates with 3 micrograms of endotoxin-free plasmids (Endofree Mini Plasmid Kit II, Tiangen, Beijing, China) using Lipofectamine 3000 (Thermo Fisher, Shanghai, China) according to the manufacturers’ guidelines. For each plasmid, transfections were carried out at least three times.
6
Generation of Polycistronic Piggyback Vectors
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The PiggyBac vector containing the coding sequence of Red Fluorescent Protein (DsRed), the neomycin resistance gene (neo) and the promoter IE2 driven Gal4 expression cassette was inserted into piggyBac vector to generate piggyBac[IE2-DsRed+IE2-Neo+IE2-Gal4]. The PiggyBac vector containing the coding sequence of Red Fluorescent Protein (DsRed), the neo and promoter 39K driven Gal4 expression cassette was used to generate piggyBac[IE2-DsRed+IE2-Neo+39K-Gal4]. The PiggyBac vector containing the Red Fluorescent Protein fused with the neo via P2A and Gal4 expression cassette was used to generate piggyBac[IE2-DsRed-P2A-Neo+39K-Gal4]. piggyBac[IE2-DsRed+IE2-Neo+IE2-Gal4], piggyBac[IE2-DsRed+IE2-Neo+39K-Gal4], and piggyBac[IE2-DsRed-P2A-Neo+39K-Gal4] were extracted from the DH5α cells with the EndoFree Mini Plasmid Kit II (TIANGEN, Beijing, China). BmN cells were transfected with 2 μg of this plasmid and a helper plasmid using X-tremeGENE HP DNA Transfection Reagent (Roche, Basel, Switzerland), and the culture medium was changed after 6 h. Three days later, the cells were cultured in Grace’s medium (Thermo Fisher Scientific, Waltham, MA, USA) containing G418 (200 μg/mL) (Merck, Darmstadt, Germany), and the culture medium was changed once every 4 d. The screening lasted for two months.
7
Molecular Detection of Feline Viruses
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pMD-FCV-F/R and pMD-FHV-1-F/R are primers designed from the genetically conserved regions of FCV VP1 and FHV-1 gB with Primer Premier 5.0 software (Table 1 ). The composition of the PCR mixture included 10 µL of 2× Taq PCR Star Mix (GenStar, China), 1 µL of each of the F/R primers (10 µmol/L), 1 µL of sample DNA/cDNA, and 7 µL of double-distilled water (ddH2O). FCV and FHV-1 were amplified using the following procedure: predenaturation at 95 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 25 s, annealing at 54 or 55 °C for 25 s, and extension at 72 °C for 30 s, with a final extension step at 72 °C for 5 min. The generated fragments were resolved on a 1.5% (W/V) agarose gel. Then, PCR products were purified using FastPure Gel DNA (Vazyme, China) and cloned into a pMD18-T vector (Takara, Japan), which resulted in the construction of two recombinant plasmids named pMD18T-FCV and pMD18T-FHV-1. Plasmids were extracted from bacterial suspensions containing accurate gene sequences using the EndoFree Mini Plasmid Kit II (Tiangen, China). The concentrations of the recombinant plasmids pMD- FCV and pMD- FHV-1 were measured, and then the numbers of copies were calculated.
8
Cloning and Characterization of IGFBP5 Vector
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The IGFBP5 vector is synthesized by Generay Bioengineering Co., Ltd. (Shanghai, China) The target fragment sequence referred to the IGFBP5 mRNA sequence (NM_001129733.1) registered on NCBI (https://www.ncbi.nlm.nih.gov/ (accessed on 10 January 2021)). The CDS region of the gene is 816 bp long, Kozak sequence was added to the 5′ end of the CDS region as well.
The successfully constructed plasmid was sent to Beijing Tsingke Biotechnology Co., Ltd. (Nanjing, China) for sequencing and comparison. At the same time, we used EndoFree Mini Plasmid Kit II (Tiangen, Beijing, China) to extract the plasmid in the remaining bacterial solution for cell transfection according to its instructions. The successfully constructed vector was named pIGFBP5-O.
The successfully constructed plasmid was sent to Beijing Tsingke Biotechnology Co., Ltd. (Nanjing, China) for sequencing and comparison. At the same time, we used EndoFree Mini Plasmid Kit II (Tiangen, Beijing, China) to extract the plasmid in the remaining bacterial solution for cell transfection according to its instructions. The successfully constructed vector was named pIGFBP5-O.
9
PiggyBac Plasmid Extraction and Transfection
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The PiggyBac[A3-EGFP+A3-Neo+IE1-scFvG4] was extracted from the DH5α cells with the EndoFree Mini Plasmid Kit II (TIANGEN, China). Sf9-III cells were transfected with 2 μg of this plasmid and a helper plasmid using X-tremeGENE HP DNA Transfection Reagent (Roche, Switzerland), and the culture medium was changed after 6 h. Three days later, the cells were cultured in Sf-900 III SFM (Thermo Fisher Scientific, Waltham Mass USA) containing G418 (200 μg/mL) (Merck, Germany), and the culture medium was changed once every 3 d. The screening lasted for two months [26 (link), 27 ].
10
Cloning and Transformation of CgTLR-TIR-BD
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A CgTLR gene sequence was obtained from GenBank (GenBank accession number: KC700619 ) and used to design original 16-bp primers. The primers used to amplify the toll/interleukin-1 receptor (TIR) domain of CgTLR were CgTLR-TIR-BD-F and CgTLR-TIR-BD-R. All of the primers used in this study are listed in Table S1 . The empty pGBK-T7 plasmid was digested by both BamHI and EcoRI and then fused with a purified CgTLR-TIR-BD PCR product using a Ligation-Free Cloning System according to the manufacturer's instructions (ABM, Inc., Ontario, Canada). The recombinant plasmid was transformed into Trans-T1 Phage Resistant Chemically Competent Cells (TransGen, Beijing, China) and extracted with an EndoFree Mini Plasmid Kit II (Tiangen, Beijing, China).
The correctly constructed CgTLR-TIR-BD plasmid was transformed into yeast strain Y2HGold, as well as empty pGBK-T7 as a control, according to the instructions of the Yeastmaker™ Yeast Transformation System 2 (Clontech). We then used 100 μl of a 1/100 dilution to coat the selective media SD/–Trp, SD/–Trp/X-α-Gal, and SD/–Trp/AbA/X-α-Gal. The toxicity of the bait protein expression plasmid CgTLR-TIR-BD was determined based on growth of bacterial colonies on the plates.
The correctly constructed CgTLR-TIR-BD plasmid was transformed into yeast strain Y2HGold, as well as empty pGBK-T7 as a control, according to the instructions of the Yeastmaker™ Yeast Transformation System 2 (Clontech). We then used 100 μl of a 1/100 dilution to coat the selective media SD/–Trp, SD/–Trp/X-α-Gal, and SD/–Trp/AbA/X-α-Gal. The toxicity of the bait protein expression plasmid CgTLR-TIR-BD was determined based on growth of bacterial colonies on the plates.
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