HEK 293 cells were seeded on tissue culture treated 24-well plates one day prior to transfection (5 × 104 cells/well). Cells were transfected using FuGENE HD transfection reagent (Promega) in fibroblast culture medium with 500 ng of either dCas9-DUX4-C or dCas9VP192 transactivator encoding plasmid and 200 ng of guide RNA-PCR product or TdTomato guide RNA plasmid. Cells were cultured for 72 h post-transfection, after which samples were collected for qRT-PCR.
Fugene hd transfection
Manufactured by Promega
Sourced in United States
FuGENE HD is a transfection reagent designed for efficient and reproducible delivery of DNA, RNA, and other macromolecules into a variety of cell types. It facilitates high-efficiency transfection with low cytotoxicity.
Automatically generated - may contain errors
Lab products found in correlation
Prl tk, by Promega (2 mentions)
Dual luciferase assay kit, by Promega (2 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
Hygromycin b, by Thermo Fisher Scientific (1 mentions)
Puromycin, by Merck Group (1 mentions)
Polybrene, by Merck Group (1 mentions)
Psmd2 g, by Addgene (1 mentions)
Tcs sp5, by Leica (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
Lysis buffer, by Promega (1 mentions)
Quick plasmid miniprep kit, by Thermo Fisher Scientific (1 mentions)
Rabbit anti β actin antibody, by Santa Cruz Biotechnology (1 mentions)
M mlv rt reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
β galactosidase enzyme assay system, by Promega (1 mentions)
Accutase, by Thermo Fisher Scientific (1 mentions)
14 protocols using fugene hd transfection
1
Transcriptional Regulation of dCas9-Mediated Targets
2
Luciferase Assay for Hedgehog and Inflammatory Signaling
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For the luciferase assay, cells in 6-well plates were co-transfected with 10 ng pRL-TK (Promega, USA) and 500 ng pGL3-8×Gli1 binding site (BS) luciferase reporter plasmids (wild-type, WT or mutant-type, MT) by FuGENE HD Transfection (Promega, USA). After 8 h, the medium was changed to 1% FBS-DMEM (control), 100 ng/mL Shh, 5 ng/mL IL-1β, or 10 ng/mL TNF-α in 1% FBS-DMEM for treatment. Luciferase assays were performed 48 h later according to the protocol of the Dual Luciferase Assay Kit (Promega, USA). The luciferase activities were normalized to the Renilla luciferase activity. MT pGL3-8×Gli1 BS luciferase report plasmids were used as control. pGL3-8×Gli1 BS luciferase reporter plasmids (WT or MT) were kindly gifted by Doctor Lei Li from the Shanghai Changhai Hospital.
3
Stable Transgenic hPSC Generation
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Stable transgenic hPSCs expressing candidates were generated by transfecting cells with PB transposon plasmids with PB transposase expression vector pBase. In order to generate the PB plasmids, the candidates (NANOG, ZNF398, KLF7, MYC, ETS2, OTX2, ID1, BCOR and PRDM14) were amplified from cDNA and cloned into a pENTR2B donor vector. Then, the transgenes were Gateway cloned into the same destination vector containing PB-CAG-DEST-bghpA and pGK-Hygro selection cassette.
For DNA transfection, 250,000 hPSCs were dissociated as single cells with TrypLE (Gibco 12563-029) and were co-transfected with PB constructs (550 ng) and pBase plasmid (550 ng) using FuGENE HD Transfection (Promega E2311), following the protocol for reverse transfection. For one well of a 12-well plate, we used 3.9 μl of transfection reagent, 1 μg of plasmid DNA, and 250,000 cells in 1 ml of E8 medium with 10 µM Y27632 (ROCKi, Rho-associated kinase (ROCK) inhibitor, Axon Medchem 1683). The medium was changed after overnight incubation and Hygromycin B (200 μg/ml; Invitrogen 10687010) was added after 48 h. For the overexpression experiments, hPSCs stably expressing an empty vector or the candidates were plated. The next day, cells were treated with DMSO or 10 µM SB43 for 5 days and then analysed as indicated in Supplementary Fig.2a .
For DNA transfection, 250,000 hPSCs were dissociated as single cells with TrypLE (Gibco 12563-029) and were co-transfected with PB constructs (550 ng) and pBase plasmid (550 ng) using FuGENE HD Transfection (Promega E2311), following the protocol for reverse transfection. For one well of a 12-well plate, we used 3.9 μl of transfection reagent, 1 μg of plasmid DNA, and 250,000 cells in 1 ml of E8 medium with 10 µM Y27632 (ROCKi, Rho-associated kinase (ROCK) inhibitor, Axon Medchem 1683). The medium was changed after overnight incubation and Hygromycin B (200 μg/ml; Invitrogen 10687010) was added after 48 h. For the overexpression experiments, hPSCs stably expressing an empty vector or the candidates were plated. The next day, cells were treated with DMSO or 10 µM SB43 for 5 days and then analysed as indicated in Supplementary Fig.
4
Stable HIF-1α Overexpression via Transfection
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HIF-1α overexpression was achieved by transfection with pcDNA3 HIF-1α-HA (Addgene, Cambridge, MA, USA) using Fugene® HD Transfection (Promega, Madison, WI, USA) according to the manufacturer's recommended protocol. After transient transfection for 24 h, cells were cultured for 4 weeks. HIF-1α positive cell clones were selected and expanded.
5
Silencing FUCA-1 Gene Expression
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FUCA-1-specific shRNA expression vector (#TG312904) and a scrambled control vector (#TR30013) were purchased from OriGene Technologies, (Inc.9620 Medical Center Drive, Suite 200 Rockville). Experimental procedures were done as follows: the day before transfection, cells were plated in 35-mm dishes at 40% of confluence in DMEM supplemented with 10% FBS without antibiotics. Two μg of sh-FUCA-1 and control vector were transfected using FuGENE HD transfection (Promega) reagent according to manufacturer's instructions. Seventy-two hours after transfection, culture medium was supplemented with puromycin (Sigma-Aldrich) at final concentration of 1μg/μl for 14 days. Stably transfected cells were screened by RT-PCR as previously described.
6
Luciferase Assay of GLI1 Binding
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The cells were seeded in six-well plates (2×105 cells per well) for 12 h and then co-transfected with 10 ng pRL-TK (Promega, USA) and 500ng pGL3-8×GLI1 binding site (BS) luciferase report plasmids (wild or mutant) by FuGENE HD transfection (Promega, USA) according to the instructions. Six hours later, the cultures were changed. Luciferase assays were performed 48 h later with the dual luciferase assay kit (Promega, USA) according to the protocol. The luciferase activities were normalized to the Renilla luciferase activity. Mutant pGL3-8×GLI1 BS luciferase report plasmids were used as a negative control.
7
Lentivirus Production and Chlamydia Infection
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For lentivirus production, 293FT cells were transfected with corresponding vectors using FuGene HD transfection (Promega, USA), following the manufacturer’s instructions. Packaging vectors were psPAX.2 and psMD2.G (Addgene Plasmids, #12260 and #12259; Dr Didier Trono). Virus-containing supernatant was collected, filtered and incubated with HeLa cells in the presence of 1 μg/ml of polybrene (Millipore, #TR-1003-G).
Chlamydia trachomatis serovar L2 (Ctr) was obtained from the American Type Culture Collection and propagated in HeLa cells. Ctr strains deficient in expression ofc hlamydial p rotease/proteasome-like a ctivity f actor (CPAF) (Rst17) and the isogenic, CPAF competent strain (Rst5) [35 (link)] were a kind gift from Dr Raphael Valdivia (Duke University School of Medicine, Durham, North Carolina, USA). Bacteria were purified over a Gastrografin density gradient (Bayer Vital, Leverkusen), followed by titration on HeLa cells and stored in SPG medium (0.2 M sucrose, 8.6 mM Na2HPO4, 3.8 mM KH2PO4, 5 mM glutamic acid [pH 7.4]) at −80 °C. Fresh aliquots were thawed for each experiment. Cells were infected at a multiplicity of infection (MOI) of 5 in complete culture medium.
Chlamydia trachomatis serovar L2 (Ctr) was obtained from the American Type Culture Collection and propagated in HeLa cells. Ctr strains deficient in expression of
8
Visualizing LvFoxP Protein Expression in Drosophila S2 Cells
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The coding sequence of LvFoxP was cloned into the pAc5.1-GFP vector (23 (link)) and transfected into Drosophila S2 cells at a confluence of ~80% using FuGENE HD Transfection (Promega, USA) to express green fluorescent protein (GFP)-tagged LvFoxP protein. At 24 h later, cells were stained with Hoechst 33342 (Sigma, USA) and visualized under a confocal laser scanning microscope (Leica TCS-SP5, Germany).
9
Cloning and Mutagenesis of STAT3 Transcription Factor
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The full-length human STAT3 was amplified by RT-PCR from the total RNA obtained from AGS cells with primers 5′-ACG CTC GAG TTA TGG CCC AAT GGA ATC AG-3′ (forward) and 5′- ATT CTT ATG CGG CCG CTC ACA TGG GGG AGG TAG-3′(reverse) and subcloned into HA-tagged pCMV expression vector (addgene Plasmid #28023) as XhoI/NotI fragment. pCMV-HA-STAT3 was used as a template. STAT3 mutant constructs were generated using a site-directed mutagenesis kit (iNtRon; Seongnam, South Korea). Primer sequences used for PCR are as follows. Y705F-FP 5′- GTA GCG CTG CCC CAT TCC TGA AGA CCA AGT TT-3′ (forward) and Y705F-RP 5′-AAA CTT GGT CTT CAG GAA TGG GGC AGC GCT AC-3′(reverse). S727A-FP 5′-GAC CTG CCG ATG GCC CCC CGC ACT TTA GAT-3′ (forward) and S727A-RP 5′-ATC TAA AGT GCG GGG GGC CAT CGG CAG GTC-3′(reverse). Mutant constructs were confirmed by DNA sequence analysis. The plasmids were transfected into AGS cells using the FuGENE HD Transfection (Promega; UK), according to the optimized protocol for the AGS cells.
10
Generation of ISKNV ORF022L Deletion Mutant
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Cells were inoculated into six-well cell culture plates and transfected with ISKNV ORF022L transfer vector by using the FuGENE HD transfection reagent (Promega, USA). At 24 h post-transfection, the cells were infected with WT ISKNV at a multiplicity of infection (MOI) of 0.02 to generate recombinant viruses. After five blind passages of puromycin (at a concentration of 2 μg/mL) selection, the red fluorescent plaques formed by recombinant viruses were picked out under an inverted fluorescence microscope, and these processes were repeated three times to obtain the purified recombinant viral strain, which was named Δ ORF022L. The thorough replacement of ORF022L by the tag genes was confirmed via PCR by using ORF022L outer primers (listed in Table S1 ), and resequencing was performed to further prove the purification of Δ ORF022L. For stability analysis, Δ ORF022L was passaged in MFF-1 cells for 10 generations, and the genome of Δ ORF022L was extracted for PCR determination by using ORF022L outer primers.
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