In order to target enhancer region efficiently which long around 1
kb, we used three sgRNA in one vector system (Adamson et al., 2016 (link)). In order to select cells infected both
sgRNA targeting PVT1 and enhancer, lentiviral vector
backbone was modified by replacing Puromycin-mCherry cassette to Hygromycin
resistance gene. Three sgRNAs were designed for each enhancer and cloned
into pMJ114 (bovine U6, Addgene, Cat#85995), pMJ117 (human U6,
Addgene, Cat#85997) or pMJ179 (mouse U6, Addgene, Cat#85996)
individually. Then, U6 promoter and sgRNA sequences were amplified by PCR
and combined into lentiviral vector using NEBuilder Hifi DNA Assembly Master
Mix, which digested with XbaI and XhoI. Cells infected lentivirus containing
sgRNA targeting PVT1 or LacZ (control)
first followed by Puromycin selection for 4 days as described above. Then,
cells were re-plated by 1:8 ratio to 6 well plate and infected with
lentivirus harboring three sgRNAs targeting each enhancer and selected with
Hygromycin for 8 days. After one day recovery without antibiotics, RNAs were
extracted and used for qRT-PCR. Sequences of sgRNAs and primers are listed
inTable S1 .
kb, we used three sgRNA in one vector system (Adamson et al., 2016 (link)). In order to select cells infected both
sgRNA targeting PVT1 and enhancer, lentiviral vector
backbone was modified by replacing Puromycin-mCherry cassette to Hygromycin
resistance gene. Three sgRNAs were designed for each enhancer and cloned
into pMJ114 (bovine U6, Addgene, Cat#85995), pMJ117 (human U6,
Addgene, Cat#85997) or pMJ179 (mouse U6, Addgene, Cat#85996)
individually. Then, U6 promoter and sgRNA sequences were amplified by PCR
and combined into lentiviral vector using NEBuilder Hifi DNA Assembly Master
Mix, which digested with XbaI and XhoI. Cells infected lentivirus containing
sgRNA targeting PVT1 or LacZ (control)
first followed by Puromycin selection for 4 days as described above. Then,
cells were re-plated by 1:8 ratio to 6 well plate and infected with
lentivirus harboring three sgRNAs targeting each enhancer and selected with
Hygromycin for 8 days. After one day recovery without antibiotics, RNAs were
extracted and used for qRT-PCR. Sequences of sgRNAs and primers are listed
in