ShRNAs targeting circPGR were cloned into lenti-viral pLKO.1 vector with AgeI and EcoRI restriction enzymes. (shRNA targeting sequence: AATCATTGCCAGGGCAGCACA (sh-circPGR#1); ATTGCCAGGGCAGCACAACTA (sh-circPGR#2)).

Full length circPGR was cloned in pCD2.1-ciR vector (Geneseed, Guangzhou) with KpnI and BamHI restriction enzymes for overexpression. Forward primer (F): 5'-GCCAAAGAATCCTGGGAGAT-3'; Reverse primer (R): 5'-CTGGCAATGATTTAGACCAC-3'. Linear sequence of circPGR (circPGR (WT)) as well as its mutant form with the potential miR-301a-5p binding site mutated (circPGR (MT)) were cloned into psiCHECK2 vector with NotI and XhoI restriction enzymes, which were named as circPGR (WT)-luc and circPGR (MT)-luc, respectively. Forward (F) primer: 5'-GGCAGCACAACTACTTAT-3'; Reverse (R) primer: 5'-CTGGCAATGATTTAGACC-3'. The primer used to mutate miR-301a-5p binding site was GTGCTCACAAACATCGTTTACATGAACTTTTTAA. 3' untranslated region (3' UTR) of CDK6, CDK1 or CHEK2 as well as the corresponding mutant form with the predicted miR-301a-5p binding site mutated were cloned into psiCHECK2 vector with NotI and XhoI restriction enzymes, which were named as 3' UTR (WT)-luc and 3' UTR (MT)-luc, respectively. CDK6-3' UTR (WT)-luc: forward primer: 5'-CAGTCTGAACCCCATTTG-3'; reverse primer: 5'- AGCTTAGCGCCTGAGAGA-3'. The primer used to mutate miR-301a-5p binding site was: TGCTTTCCGAGTCGTAGTTGCCCTGCTGCTGTCT. CDK1-3' UTR (WT)-luc: forward primer: 5'-CATCAGTTTCTTGCCATGT-3'; reverse primer: 5'- GAGCCTTTTTAGATGGCTG-3'. The primer used to mutate miR-301a-5p binding site was: AGTGAATTCTTATGCCTTGTCGTAGTTAATAACTGA. CHEK2-3' UTR (WT)-luc: forward primer: 5'-GCGCCTGAAGTTCTTGTT-3'; reverse primer: 5'- TGGGGTAGAGCTGTGGAT-3'. The primer used to mutate miR-301a-5p binding site was AAGTCTGGGCAGAAGTCCGTAGTAAAGCTCTGGACC.

Carboxyl (C)-terminal 3×Flag-tagged AGO2 was cloned into pCDH-CMV-puromycin vector with NotI and BamHI restriction enzymes: forward: 5'-ATGTACTCGGGAGCCGGC-3'; reverse: 5'-TCAAGCAAAGTACATGGT-3'.

注意:以上内容是从某篇研究文章中自动提取的,可能无法正确显示。



Q&A
请登录并在线提交您的问题
您的问题将发布在Bio-101网站上。我们会将您的问题发送给本研究方案的作者和具有相关研究经验的Bio-protocol成员。我们将通过您的Bio-protocol帐户绑定邮箱进行消息通知。