Cloning of SMPD3 and SMPD4 cDNA

cDNA encoding SMPD3 and SMPD4 were kindly donated by Dr. Hannun [15 (link)] and Dr. Mangini, respectively [12 (link)]. 3xHA-miniTurbo-NLS_pCDNA3 was a gift from Alice Ting (Addgene plasmid # 107172; http://n2t.net/addgene:107172 (accessed on 31 October 2018); RRID:Addgene_107172). Ref. [16 (link)]. All steps of the cloning process were performed according to the protocol provided by the manufacturer. To enable cloning, restriction enzyme recognition sites were introduced into the SMPD3 and SMPD4 cDNA using overhanging primers in a 2-step PCR (Appendix B Table A2). Vectors were digested using enzymatic restriction digestion, dephosphorylated with Shrimp Alkaline Phosphatase (New England Biolabs, Ipswich, MA, USA), and ligated to purified PCR products using T4 DNA ligase (NEB). Plasmids were introduced into One Shot Stbl3 chemically competent E. coli (Thermo Fisher Scientific Inc., Waltham, MA, USA). For miniprep, a colony was inoculated and grown in Luria–Bertani medium with 50 µg/µL of ampicillin, and plasmid DNA was isolated using a QIAprep Spin Miniprep Kit (Qiagen, Venlo, The Netherlands). For midiprep, DNA was isolated using the NucleoBond Xtra Midi kit (Bioké, Leiden, The Netherlands). DNA fragments amplified with PCR were sequenced by the Macrogen EZ-Seq service (Macrogen, Amsterdam, The Netherlands).