sgRNAs targeting indicated AR promoter regions (Table S5 ) were designed using
the MIT CRISPR Design software (crispr.mit.edu ). Each sgRNA oligos were synthesized and
cloned into lentiCRISPR v2 vector as a gift from Dr. Feng Zhang (Addgene
pladmid #52961). Lentiviral particles was produced in 293T with PEI
transfecting reagent (VWR). LNCaP cells were then infected with sgRNAs
lentiviral particles combination for 48 hours, then split, and transfected
with either control or siEZH2 using Lipopectamine 2000 (Invitrogen) for 48
hours. Genomic DNA was prepared using the PureLink Genomic DNA kit (Life
Technology). PCR of genomic DNA was performed with indicated primers
flanking the sgRNA target sites on AR promoter region (Table S5 ). PCR products were
purified from agarose gel and sequenced to assess the effects of
CRISPR-Cas9-mediated editing of AR promoter. Total RNA was isolated from
cells with Nucleospin RNA isolation kit (Clonetech) and 250 ug of RNA per
sample was used for cDNA synthesis using qscript cDNA synthesis supermix
(Quantabio). PCR of cDNA were then performed using specific AR promoter
(also exon 1) primers (Primer F2 and R2) and subjected for agarose gel
analysis. Protein extracts were subjected for western blot analysis to
confirm EZH2 knockdown.
the MIT CRISPR Design software (
cloned into lentiCRISPR v2 vector as a gift from Dr. Feng Zhang (Addgene
pladmid #52961). Lentiviral particles was produced in 293T with PEI
transfecting reagent (VWR). LNCaP cells were then infected with sgRNAs
lentiviral particles combination for 48 hours, then split, and transfected
with either control or siEZH2 using Lipopectamine 2000 (Invitrogen) for 48
hours. Genomic DNA was prepared using the PureLink Genomic DNA kit (Life
Technology). PCR of genomic DNA was performed with indicated primers
flanking the sgRNA target sites on AR promoter region (
purified from agarose gel and sequenced to assess the effects of
CRISPR-Cas9-mediated editing of AR promoter. Total RNA was isolated from
cells with Nucleospin RNA isolation kit (Clonetech) and 250 ug of RNA per
sample was used for cDNA synthesis using qscript cDNA synthesis supermix
(Quantabio). PCR of cDNA were then performed using specific AR promoter
(also exon 1) primers (Primer F2 and R2) and subjected for agarose gel
analysis. Protein extracts were subjected for western blot analysis to
confirm EZH2 knockdown.