E coli mach1 chemically competent cells

Yeast expression plasmids carrying human HINT1 (pAG415-HINT1-hWT & pAG415-HINT1-Arg37Pro) were generated in a previous study [1 (link)]. Mammalian expression plasmid carrying human HINT1 (pCAGGS-HINT1-hWT) was created at the VIB Protein Service Facility (UGent, Ghent, BE). The different HINT1 variants were introduced with site directed mutagenesis using KAPA HiFi DNA polymerase (Roche Diagnostics, Basel, CH). After overnight DpnI digestion (New England Biolabs, Ipswich, MA) products were transformed into E. coli Mach1 chemically competent cells (ThermoFisher Scientific, Waltham, MA, USA) and validation of the correct incorporation of the missense variant was done by Sanger sequencing of the purified plasmid.

Total RNA was isolated from lymphoblasts using the Universal RNA kit (Roboklon, Berlin, GE) and treated with the Turbo DNA-free kit (Ambion, Austin, TX, USA) to remove residual gDNA. RNA was transcribed to single strand cDNA with the iScript cDNA advanced synthesis kit (Bio-Rad, Hercules, CA, USA). HINT1 cDNA was amplified using HINT1-specific primers with overhangs containing restriction sites for EcoRI (forward primer) and HindIII (reverse primer) and introduced into a pUC19 plasmid after double digestion with FastDigest-EcoRI and FastDigest-HindIII (ThermoFisher Scientific, Waltham, MA, USA) followed by ligation with T7 DNA ligase (Enzymatics, Beverly, MA, USA). Products were transformed into E. coli Mach1 chemically competent cells (ThermoFisher Scientific, Waltham, MA, USA) and plated on ampicillin agar. Positive colonies were picked and grown overnight in 5 ml LB broth supplemented with 100 µg/ml ampicillin. Cells were harvested and plasmid was purified using the NucleoSpin Plasmid EasyPure kit (Macharey-Nagel, Bethlehem, PA, USA). Phasing of the variants was performed by Sanger sequencing of individual clones.