Human placenta genomic dna

Minigene fragments were amplified from human placenta genomic DNA (Sigma) (for intronic sequences) or EC109 cDNA (for exonic sequences) using PrimeSTAR Max DNA polymerase (Clontech) with overlapped primers and ligated into one piece of DNA, followed by ligation into pcDNA3.1+ vector. KAPA HiFi polymerase (KAPA Biosystems) was used to introduce point mutations into minigene using primers with corresponding mutation(s).
Overexpression plasmids were obtained by cloning coding sequences of protein, which were amplified by PrimeSTAR Max DNA polymerase (Clontech), into pLenti6 vector. ADARs-targeting short hairpin RNAs (shRNAs) were designed using RNAi Platform (Broad Institute) and were cloned into pLKO.1_puro plasmid using AgeI and EcoRI restriction sites.
CasRX system (pXR001: EF1a-CasRx-2A-EGFP and pXR003: CasRx gRNA cloning backbone) was a gift from Patrick Hsu (pXR001: Addgene plasmid #109049, http://n2t.net/addgene:109049, RRID:Addgene_109049; pXR003: Addgene plasmid #109053, http://n2t.net/addgene:109053, RRID:Addgene_109053)^{51 (link),52} . Guide RNA (gRNA) sequences were designed using sequence of circRNAs around BSJ with a length of 21 bp and cloned into pXR003 plasmid using BbsI restriction sites.

Minigene sequences were amplified from human placenta genomic DNA (Sigma) using PrimeSTAR Max DNA polymerase (Clontech) and subcloned into pcDNA3.1 + plasmid using BamHI and XhoI restriction sites. Deletions and point mutations were introduced into minigene plasmid by PCR mutagenesis using KAPA HiFi polymerase (KAPA Biosystems) with primers containing respective mutations. Coding sequences of protein were amplified by PrimeSTAR Max DNA polymerase (Clontech) and cloned into pLenti6 vector with respective restriction sites. shRNA sequences were designed using RNAi Platform (Broad Institute) and cloned into pLKO.1_puro plasmid using AgeI and EcoRI restriction sites.