Lentiviral Vector Transduction of PCa Cells

Lenti dCAS-VP64_Blast (Addgene plasmid # 61425; http://n2t.net/addgene:61425;
RRID:Addgene_61425), lenti MS2-P65-HSF1_Hygro (Addgene plasmid # 61426;
http://n2t.net/addgene:61426;
RRID:Addgene_61426), and lenti sgRNA(MS2)_puro backbone (Addgene plasmid #
73795; http://n2t.net/addgene:73795;
RRID:Addgene_73795) were a gift from Feng Zhang. Nontargeted and
ARID5A guide RNA (gRNA) sequences used in this study are
shown in Supplemental Table
S2. The top and bottom strand oligonucleotides were annealed, and
inserted into lenti sgRNA(MS2)_puro backbone vector according to the
reference.^{25 (link)}Lentiviral particles were produced in 293T cells transfected with lenti
dCAS-VP64_Blast, lenti MS2-P65-HSF1_Hygro, or lenti sgRNA(MS2)_puro encoding
guide RNAs, and lentiviral packaging plasmids (BioSettia; San Diego, CA) using
Lipofectamine 3000 reagent. The culture supernatants were collected and
concentrated using Speedy Lentivirus Purification reagent (Applied Biological
Materials, Inc.; Richmond, British Columbia, Canada). The viral titer was
determined using 293T cells as described previously.²⁶ PCa cells were transduced for 24 hr at
MOI’s of 1-10 in the presence of 6 μg/mL polybrene.