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Supplementary Materials and Methods Plasmid Construction


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Supplementary Materials and Methods
Plasmid Construction

For generating lentiviral vectors expressing TuD RNA against let-7a or a negative control under the human 7SK RNA polymerase III promoter (1), pLSP-TuD-let7a and pLSP-TuD-NC were constructed by inserting an annealed complementary pair of oligonucleotides (5′-cat caa cta tac aac caa tgt act acc tca cgt att ctg gtc aca gaa tac aac tat aca acc aat gta cta cct cac g-3′ and 5′-tca tcg tga ggt agt aca ttg gtt gta tag ttg tat tct gtg acc aga ata cgt gag gta gta cat tgg ttg tat agt t-3′ for let-7a, and 5′-CAT CAA CAA GCC ACA ACG AAT CTC TAT ATC ATC AAG TAT TCT GGT CAC AGA ATA CAA CAA GCC ACA ACG AAT CTC TAT ATC ATC AAG-3′ and 5′-TCA TCTT GAT GAT ATA GAG ATT CGT TGT GGC TTG TTG TAT TCT GTG ACC AGA ATA CTT GAT GAT ATA GAG ATT CGT TGT GGC TTG TT-3′ for NC) as described previously (2).



For generating B5R-modified recombinant viruses, we amplified the B4R, B5R, and B6R genes with primers 5′-TCG GAA GCA GTC GCA AAC AAC-3′ and 5′-ATA CCA TCG TCG TTA AAA GCG C-3′ using LC16mO genomic DNA as a template. The PCR product was cloned into the pCRII plasmid (Invitrogen) to generate pB5R. The DNA fragment carrying NheI and AgeI sites at the 3′UTR of the B5R gene was generated by PCR amplification, with pB5R as the template, using the following primers: 5′-CAA ACT CTC GAA AGA CGT-3′ and 5′-gca ccg gtg cta gcT TAC GGT AGC AAT TTA TGG AA-3′, or 5′-ccg cta gca ccg gtA TAT AAA TCC GTT AAA ATA ATT AAT-3′ and 5′-CAG GAA ACA GCT ATG AC-3′ (M13 reverse primer; Invitrogen). The generated PCR products were digested with HpaI and NheI or NheI and HindIII, respectively, and were subcloned into the HpaI and HindIII sites of pB5R by three-part ligation, resulting in pTN-B5R. Two oligonucleotides containing two copies of completed or mutated complementary target sequences for let-7a miRNA plus the MluI site (5′-cta gcA ACT ATA CAA CCT ACT ACC TCA cga tAA CTA TAC AAC CTA CTA CCT CAc gcgt a-3′ and 5′-ccg gta cgc gTG AGG TAG TAG GTT GTA TAG TTa tcg TGA GGT AGT AGG TTG TAT AGT Tg-3′ or 5′-cta gcA ATT ACA CGA CTT ATT ATT TGA cga tAA TTA CAC GAC TTA TTA TTT GAc gcg ta-3′ and 5′-ccg gta cgc gTC AAA TAA TAA GTC GTG TAAT Tat cgT CAA ATA ATA AGT CGT GTA ATT g-3′) were annealed and subcloned into pTN-B5R at the corresponding restriction sites NheI and AgeI, resulting in pTN-B5Rlet7a ×2 and pTN-B5Rlet7a-mut ×2, respectively. Another pair (5′-cgc gtA ACT ATA CAA CCT ACT ACC TCA tca cAA CTA TAC AAC CTA CTA CCT CA-3′ and 5′-ccg gTG AGG TAG TAG GTT GTA TAG TTg tga TGA GGT AGT AGG TTG TAT AGT Ta-3′ or 5′-cgc gtA ATT ACA CGA CTT ATT ATT TGA tca cAA TTA CAC GAC TTA TTA TTT GA-3′ and 5′-ccg gTC AAA TAA TAA GTC GTG TAA TTg tga TCA AAT AAT AAG TCG TGT AAT Ta-3′) was annealed and cloned into pTN-B5Rlet7a ×2 or pTN-B5Rlet7a-mut ×2 at the corresponding restriction sites MluI and AgeI, resulting in pTN-B5Rlet7a ×4 or pTN-B5Rlet7a-mut ×4, respectively. We also amplified the DNA sequence encoding the fusion protein of B5R and EGFP with primer pairs 5′-caa aat att ttc gtt gcg aag a-3′ and 5′-CAC CAT GGG TAG CAA TTT ATG GAA CT-3′ or 5′-GCG GCC GGA CCG GCC ACC ATG GTG AGC AAG GGC GA-3′ and 5′-gcg cta gcT TAC TTG TAC AGC TCG TCC A-3′ using pTN-B5R or pEGFP-N1 (Clontech) as a template. The generated PCR products were digested with HpaI and NcoI or NcoI and NheI, respectively, and were subcloned into the HpaI and NheI sites of pTN-B5R, pTN-B5Rlet7a ×4, or pTN-B5Rlet7a-mut ×4 by three-part ligation, resulting in pTN-B5Rgfp, pTN-B5Rgfplet7a ×4, or pTN-B5Rgfplet7a-mut ×4. For generating recombinant viruses expressing luciferase and EGFP, we amplified the entire region of the firefly luciferase gene with primers 5′-GCG GCC GGA CCG GCC ACC ATG GAA GAT GCC AAA AA-3′ and 5′-ATG GCC GGC CTT ACA CGG CGA TCT TGC CGC-3′ using pGL4 (Promega) as the template. The SfiI/FseI-digested PCR product was cloned into pSFJvnc110, containing the highly efficient vaccinia promoter (3) at the corresponding restriction sites, resulting in pSFJvnc110-Luc. The SmaI/NotI-digested EGFP gene from pEGFP-N1 (Clontech) was subcloned into pIRES (Clontech) at the corresponding restriction sites, resulting in pIRES-EGFP. We digested pIRES-EGFP with MluI and NotI and blunted the ends with T4 DNA polymerase. The blunted IRES-EGFP fragment was cloned into the blunted FseI site of pSFJvnc110-Luc, resulting in pSFJvnc110-LucIRESgfp.

For generating reporter plasmids containing two expression units encoding Renilla luciferase acting as a transfection control and firefly luciferase with four copies of let-7a target sequences or the disrupted sequences in the 3’UTR, we digested pTN-B5Rlet7a ×4 or pTN-B5Rlet7a-mut ×4 with AgeI and blunted the ends with T4 DNA polymerase. Furthermore, the blunted pTN-B5Rlet7a ×4 or pTN-B5Rlet7a-mut ×4 was digested with NheI, and cloned into the NheI and blunted XhoI sites of pMirGlo (Promega), resulting in pMirGlolet7a or pMirGlolet7a-mut.






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