TransIT®-LT1 Transfection Reagent

Protocol tips

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	Incubate TransIT-LT1 Reagent:siRNA complexe mixture at room temperature for 15–30 minutes to allow sufficient time for complexes to form and add to cells.  Incubate for 72 hours

Protocol tips
Incubate TransIT-LT1 Reagent:siRNA complexe mixture at room temperature for 15–30 minutes to allow sufficient time for complexes to form and add to cells.  Incubate for 72 hours

Publication protocol

Over-expression FIV lenti-vectors were purchased from GeneCopoeia for miR-142-3p (HmiR02082-MR01) and a scramble sequence control (CmiR0001-MR01) (hereafter miR-142 OE and OE Control). The miR-142 OE vector produces both 3p and 5p strands. Vectors were packaged using 293T cells and a GeneCopoeia Lenti-Pac FIV Expression Packaging Kit (FPK-LvTR) according to the manufacturer's recommendations. For knockdown of Rab27A, shRNA vectors were purchased from Dharmacon. Five vectors were purchased to determine knockdown efficiency (TRCN0000005294, TRCN0000005295, TRCN0000005296, TRCN0000005297, TRCN0000005298) the 2 vectors with the highest efficiency were used for ongoing analysis (TRCN0000005295, TRCN0000005296 hereafter 5295 and 5296 respectively). Empty pLKO.1 was used as a control. SEV staining vector pCT-CD63-GFP was purchased from SystemBio. For rescue experiments a TGFBR1 open reading frame (ORF) clone (TRCN0000488036) within a pLX_TRC317 vector as well as an empty vector control were purchased from Sigma Aldrich (hereafter TGFBR1 ORF and Control ORF). Lentivirus was created using 293T cells with each shRNA, GFP or ORF plasmid, packaging plasmids VSVG and d8.91 using TransIT-LT1 transfection reagent (Mirus). For all vectors, virus-containing conditioned media was collected over 3 days post transfection and filtered using 0.45 μm filters and stored at −80°C. Cells were selected over 10 days using either 400 μg/mL G418 (over-expression lines) or 2 μg/mL puromycin (shRNA, ORF and GFP lines).

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Manufacturer protocol

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