One control non-targeting shRNA lentivirus (ntc, thereafter referred to as control cells) and five shRNA lentiviruses directed against Pex16 (shPex16 1–5) were purchased from Sigma (MISSION® shRNA lentiviral particles NM_145122), see Table S1 . 3T3-L1 cells were seeded into 6-well plates 12 h before transduction using 3 × 104 cells/well (around 30% confluence). Cells were infected for 24h with a multiplicity of infection (MOI) of 7.5 in complete medium containing 8 μg/mL polybrene (Sigma). After transduction, the infection medium was replaced with fresh medium and cells were selected with puromycin (3 μg/mL) for 7 days. Silencing efficiency was controlled by quantitative RT-PCR. Only shRNAs #1, #4 and #5 were effective in silencing Pex16. For quantitative RT-PCR analysis and ORO stainings of the 3 efficient silencing constructs #1, #4 and #5 combined see Fig. S1D . Experiments were performed with biological replicates of 3T3-L1 cells silenced with shRNA #4 (referred to as shPex16 in the manuscript), showing the highest silencing efficiency.
Mission shrna lentiviral particles nm 145122
Manufactured by Merck Group
MISSION® shRNA lentiviral particles NM_145122 are a laboratory tool designed for targeted gene knockdown experiments. The particles contain short hairpin RNA (shRNA) sequences that are expressed within target cells to induce silencing of the gene of interest, NM_145122. This product provides a standardized and efficient method for gene function analysis in a variety of cell types.
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2 protocols using mission shrna lentiviral particles nm 145122
1
Silencing of Pex16 in 3T3-L1 Cells
2
Silencing of Pex16 in 3T3-L1 Cells
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One control non-targeting shRNA lentivirus (ntc, thereafter referred to as control cells) and five shRNA lentiviruses directed against Pex16 (shPex16 1–5) were purchased from Sigma (MISSION® shRNA lentiviral particles NM_145122), see Table S1 . 3T3-L1 cells were seeded into 6-well plates 12 h before transduction using 3 × 104 cells/well (around 30% confluence). Cells were infected for 24h with a multiplicity of infection (MOI) of 7.5 in complete medium containing 8 μg/mL polybrene (Sigma). After transduction, the infection medium was replaced with fresh medium and cells were selected with puromycin (3 μg/mL) for 7 days. Silencing efficiency was controlled by quantitative RT-PCR. Only shRNAs #1, #4 and #5 were effective in silencing Pex16. For quantitative RT-PCR analysis and ORO stainings of the 3 efficient silencing constructs #1, #4 and #5 combined see Fig. S1D . Experiments were performed with biological replicates of 3T3-L1 cells silenced with shRNA #4 (referred to as shPex16 in the manuscript), showing the highest silencing efficiency.
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