siRNA oligonucleotides targeting mouse Rubcn (CTCAGAGTAACAGGACCTT) were synthesized by OBiO Technology (Shanghai). Astrocytes were transfected with 50 nM Rubcn siRNA or the scramble control using jetPRIME® in vitro DNA & siRNA transfection reagent (Polyplus, Cat#: 114-15) according to the manufacturer’s protocol. Seventy hours after transfection, astrocytes were collected for Western blot detection and functional assays.
Jetprime in vitro dna sirna transfection reagent
Manufactured by Polyplus Transfection
Sourced in France
JetPRIME® is an in vitro DNA and siRNA transfection reagent. It facilitates the delivery of nucleic acids into eukaryotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Virapower mix, by Thermo Fisher Scientific (2 mentions)
Sirna oligonucleotides, by Obio Technology (1 mentions)
Pentr d topo, by Thermo Fisher Scientific (1 mentions)
Gateway lr clonase 2, by Thermo Fisher Scientific (1 mentions)
Puromycin, by Solarbio (1 mentions)
Pfu polymerase, by Thermo Fisher Scientific (1 mentions)
Pdsred2 mito vector, by Takara Bio (1 mentions)
Pcdna5 vector, by Thermo Fisher Scientific (1 mentions)
Pacgfp1 mito vector, by Takara Bio (1 mentions)
9 protocols using jetprime in vitro dna sirna transfection reagent
1
Silencing Rubcn in Astrocytes
2
Human Podocyte Cell Transfection and Treatment
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The human podocyte cell line was provided by M. Saleem (Saleem et al., 2002 (link)) (University of Bristol, United Kingdom) and was cultured as described previously (Wu et al., 2014 (link)). Human podocytes in 6-well plates were ready for transfection. For transfection of plasmids or siRNA, jetPRIME in vitro DNA & siRNA transfection reagent (PolyPlus Transfection, pt-114-15) were used according to the manufacturer’s instructions. In brief, 2 µg plasmid was diluted into 200 µl jetPRIME buffer, mixed by vortex. After adding 4 µl jetPRIME reagent and incubating for 10 mins, the transfection mixture was added to the cells in serum containing medium. For siRNA transfection, 110 pmole siRNA was diluted into 200 µl jetPRIME buffer, mixed by vortex. After adding 3 µl jetPRIME reagent and incubating for 10 mins, the transfection mixture was added to the cells in serum containing medium. Cells were harvest at 24 h after transfection. Cells were treated with 10 µM Adriamycin (MCE, HY-15142) for 6 h and 5 µM MG132 (MCE, HY-13259) for 1 h before harvested.
3
Generating Mafa-AG Overexpressing Macaque Cell Line
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A macTSC#2 line overexpressing Mafa-AG was established to set the threshold in flow cytometric analysis. A full-length ORF of Mafa-AG was amplified by using a primer set that added a 3xFlag tag at the N-terminus (Table S5 ) and cloned into pENTR™/D-TOPO® (Invitrogen). The 3xFlag-Mafa-AG cDNA insert was transferred into a destination vector, including Venus and puromycin-resistant genes50 (link) using Gateway® LR Clonase™ II (Invitrogen). The resulting Mafa-AG/Venus-expression vector was transfected into macTSC by using jetPRIME® in vitro DNA & siRNA Transfection Reagent (Polyplus Transfection, Illkirch, France) for 6 hours in the FHBY condition. Transfected cells were selected with 10 µg/ml puromycin for 48 hours after the transfection. Single Venus-positive colonies were picked and expanded separately in the FHBY condition. A cell line with a uniform and strong Venus fluorescence was used in the present study.
4
Plasmid Transfection and Puromycin Selection
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The cells were seeded in a 6-well plate in advance. Plasmids were transfected based on the protocol of jetPRIME® in-vitro DNA & siRNA transfection reagent (Polyplus). Jetprime buffer (200 μl), plasmid (2 μg), and jetprime (4 μl) were mixed together. The transfection mixture was incubated at a room temperature for 10 min. Then, cells in the 6-well plate were incubated with transfection mixture (200 μl per well) for 4 h, after which the media was replaced with normal media. puromycin solution (0.4 μl) (Solarbio, 10 mg/mL) was added to 2 mL medium to prepare the puromycin dilution. After the plasmid transfection, the cells in the SIRT1 shRNA group and SIRT1 shRNA Control group were selected with puromycin dilution for 24 h at CO2 incubator.
5
ATAD3A Cloning and G355D Mutagenesis
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Human ATAD3A cDNA was cloned into the pcDNA5 vector (Invitrogen) using BamHI and XhoI restriction sites. Primers used for amplification were: 5’-GCCGGATCCGGAATGTC GTGGCT CTTCGGCATT-3’, and 5’-CGCCTCGAGTCA GGAT GGGG A G GGCTCGTC-3’. Site-directed mutagenesis was conducted to introduce the G355D mutation with primers 5’-ATGTAC GGG CCACCAGACACCGGGAAGACG-3’, and 5’- CGTCT TCCCG GTG TC TGG TGGCCCGTACAT-3’ using the Pfu polymerase (Thermo Fisher). The plasmid sequences were verified by Sanger sequencing. Primary human fibroblasts were cultured in DMEM/FBS/L-Glutamine/Uridine in 37°C, 95% Oxygen, 5% CO2. Plasmid transfections to the cells were conducted according to the manufacturer’s instructions using jetPRIME in vitro DNA & siRNA transfection reagent (Polyplus). Cells for immunocytochemistry were co-transfected with mitochondria-targeted red fluorescent protein (mito-Red) (pDsRed2-Mito Vector, Clontech) or mito-GFP (pAcGFP1-Mito Vector, Clontech).
6
Establishment of macTSC Overexpressing Venus
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macTSC#2 and #4 lines overexpressing Venus was established for xenogeneic chimera assay. The 3x Flag Venus-expression vector50 (link) was transfected into macTSC using jetPRIME® in vitro DNA & siRNA transfection reagent (Polyplus Transfection, Illkirch, France) for six hours in the FHBY condition. Transfected cells were selected with 10 µg/ml puromycin for 48 hours after the transfection. Single Venus-positive colonies were selected and expanded separately in the FHBY condition.
7
Generating Stable Ehmt2 (G9a) Knockdown Cell Lines
8
Generating Stable Ehmt2 (G9a) Knockdown Cell Lines
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The lentiviral plasmids for the shRNA targeting mouse Ehmt2 (G9a) in a vector pLKO.1 were purchased from Thermo Scientific Open Biosystems. The shRNA-Ehmt2 target sequence is TTGTTATCTAAATCGAAGAGG. A pLKO.1 empty vector (EV) without shRNA sequence was used as the wild-type control. To generate virus, pLKO.1-shRNA plasmids were co-transfected into 293T cells with ViraPower Mix (Invitrogen) by jetPRIME™in vitro DNA & siRNA transfection reagent (Polyplus). Viral supernatants were collected 24 and 48 hours following transfection and were used to transduce Raw264.7 cells by spinoculation. Forty-eight hours after the transfection, 8μg/ml puromycin was added to initially select puromycin-resistant clones. The stable clones were maintained in the media containing 4 μg/ml puromycin. The level of G9a expression was examined for individual clones. Those clones with at least 90% knockdown efficiency were used in the experiments.
9
Silencing ACE2, SNX27, and VPS35 Genes for SARS-CoV-2 Pseudovirus Infection Study
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SMARTpool siRNAs targeting ACE2 (5′-GCGACUUCAGGAUCCUUAU-3′), SNX27 (5′-CGGUUACAGUCAGGGUUAA-3′), and VPS35 (5′-GCUGGCAGAAUUGCCCUUA-3′) were designed and synthesized by the supplier GenePharma Biotechnology. We selected the most efficient siRNA for each gene for the experiment. Huh7 cells were seeded in 48-well plates (2.5 × 104 cells per well) and transfected with 300 nM siRNA using jetPRIME in vitro DNA & siRNA Transfection Reagent (Polyplus Transfection, 114). Then, the cells were incubated in DMEM at 37 °C for 48 h or 72 h. The reaction mixture was discarded, and the cells were infected with SARS-CoV-2 pseudovirus and detected by Luciferase Assay System. A control siRNA (UUCUCCGAACGUGUCACGU) was maintained in our laboratory and used as a negative control. The silencing efficiency was measured by immunoblotting.
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