PCR reactions for cloning purpose used Phusion enzyme from ThermoFisher Scientific (Pittsburgh, PA, USA). Different fragments of Linc-RoR were cloned into pCDH-CMV-MSC-EF1-copGFP (System Biosciences) with a strategy described previously (13 (link)). For example, to clone Linc-RoR E4, we first amplified the entire exon 4 by PCR using primers RoR-E4-R1–5.1 and RoR-E4-Not1-3.2 (Supplementary Table S1) and then cloned into the designated vector at EcoR I and Not I sites using Cold Fusion kit (System Biosciences). To make miR-145 binding site mutant clones, we carried out a two-step amplification procedure as described previously (19 (link)), using primers RoR-miR145-BS1-m-5.1 and RoR-miR145-BS1-m-3.1; RoR-miR145-BS2-m-5.1 and RoR-miR145-BS2-m-3.1 (Supplementary Table S1). All PCR products were verified by DNA sequencing. RoR-E4 clone mutated at two putative binding sites was made through the gBlock method from IDT.
Protocol detail
Cloning Linc-RoR and miR-145 Mutants
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Corresponding Organization :
Other organizations : University of Mississippi Medical Center, Jackson Memorial Hospital, Northwestern University, Tongji University, Tongji Hospital, Zhejiang Sci-Tech University, Jiangsu University, Affiliated Hospital of Jiangsu University
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Variable analysis
independent variables
- Linc-RoR fragments cloned into pCDH-CMV-MSC-EF1-copGFP vector
- Primers used for cloning (RoR-E4-R1–5.1, RoR-E4-Not1-3.2, RoR-miR145-BS1-m-5.1, RoR-miR145-BS1-m-3.1, RoR-miR145-BS2-m-5.1, RoR-miR145-BS2-m-3.1)
- Mutations introduced in miR-145 binding sites
- Not explicitly mentioned
- Phusion enzyme from ThermoFisher Scientific used for PCR
- Cold Fusion kit (System Biosciences) used for cloning
- DNA sequencing used to verify PCR products
- Not explicitly mentioned
- Not explicitly mentioned
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