Pet28alic

PADs 1, 2, 3 and 4 were purified as reported.^{44 (link), 53 (link)} Rhodamine-phenylglyoxal (Rh-PG) was synthesized as reported.^{43 (link)} The plasmid pET28a-LIC, harboring the NNMT gene was purchased from Addgene (Addgene plasmid # 40734). All chemicals were purchased from Sigma.

We obtained the pET28aLIC (Plasmid #26094) and pL-CRISPR.efs.gfp (plasmid # 57818) plasmids from Addgene and the pLVXTRE3GZsGreen1 vector from Clontech. We amplified the coding sequence of the NT5C2 cDNA from pLOC-NT5C2 (Tzoneva et al., 2013 ) and cloned it into the pET28aLIC vector using In-fusion cloning using the In-Fusion HD Cloning Kit (Clonetch) following manufacturer guidelines. We cloned the NT5C2 S408-D415 loop deletion mutation into the pET28aLIC and pLVXTRE3GZsGreen1 vector using Gibson Assembly using the Gibson Assembly Master Mix (New England Biolabs) following manufacturer guidelines. We cloned a truncated active form of NOTCH1 ΔE-NOTCH1 (Schroeter et al., 1998 (link)) into the pMSCV-pBabeMCS-IRES-RFP retroviral vector (Addgene plasmid # 33337). We generated lentiviral vectors expressing CAS9 and gRNAs targeting the arm segment of mouse Nt5c2 by cloning the corresponding gRNA oligonucleotides (Sigma-Aldrich) into pL-CRISPR.efs.gfp as reported (Shalem et al., 2014 (link)).

We obtained the pET28aLIC (Plasmid #26094) and pL-CRISPR.efs.gfp (Plasmid #57818) plasmids from Addgene and pLVXTRE3GZsGreen1 vector from Clontech. We amplified the coding sequence of the NT5C2 cDNA from pLOC-NT5C2(7 (link)) and cloned it into the pET28aLIC and pLVXTRE3GZsGreen1 vectors using In-fusion cloning using the In-Fusion HD Cloning Kit (Clontech) following manufacturer guidelines. We generated lentiviral vectors expressing CAS9 and gRNAs targeting exon 3 or 8 of Nt5c2 by cloning the corresponding gRNA oligonucleotides (Sigma-Aldrich) into pL-CRISPR.efs.gfp as reported(42 (link)). We cloned NT5C2 R238W, K359Q, R367Q, L375F, D407A, K217R, K217Q, K344R, K344Q, S418A, S418D, S502A, S502D, D229A and D229S mutations into the pLOC-NT5C2(7 (link)) or pET28aLIC-NT5C2 by site directed mutagenesis using the QuikChange II XL Site-Directed Mutagenesis kit (Agilent Technologies) according to manufacturer’s guidelines.