Sureclean kit

A 456 bp fragment of the TEP1-TED domain was amplified with primers OB2996F (5'-CACGGTCATCAAGAACCTGGAC-3') [19 (link)] and EMTep1R (5’-TCCAGCAATGCCATCAACACAT-3'), the latter specifically designed for the aim of this work in order to avoid co-amplification of other TEP-related paralogs, which were instead pervasively co-amplified with other primer couples available in literature. Amplifications were performed in a 15 μl reaction-mix using 0.5–1.0 μl of template DNA using the High Fidelity AccuPrime Taq DNA Polymerase kit (Life Technologies) following manufacturer's guidelines. Thermocycler conditions were as follows: initial denaturation at 94°C for 2 min followed by 35 cycles of 94°C for 30 sec, 54°C for 30 sec, 68°C for 1 min, with a final elongation at 68°C for 7 min. The resulting products were analysed on 1–2% agarose gels stained with GelRed (Biotium), purified with the SureClean Kit (Bioline) and sequenced at the BMR Genomics s.r.l. (Padua, Italy). Sequences are available in GenBank under Accession Numbers KR091079—KR091309.

Error prone PCR was performed on 30 ng template plasmid for 25 cycles using GeneMorph II Random Mutagenesis Kit from Agilent Technologies. To clone the libraries, 400 ng of digested PCR product was ligated to 400 ng of digested pUCBAD-Kan at 16°C overnight using T4 DNA ligase. The ligation mixture was purified with SureClean Kit (Bioline) and dissolved in 10 µl of water. The ligated DNA was transformed into MegaX DH10B T1R Electrocomp Cells (Life Technologies) by electroporation. All the MegaX DH10B T1R transformants were subsequently propagated for 24 hrs at 37°C in 20 ml LB medium with kanamycin. Plasmid DNA was prepared from the overnight culture and used to transform the Salmonella typhimurium LT2 strain by electroporation.

SMCs were transfected with lentiviral vector encoding reprogramming factors Oct4, Sox2, Klf4 and c-Myc (TetO-FUW-OSKM, Addgene Plasmid 20321) or control empty vector and maintained in reprogramming media consisting of Knockout DMEM/F12 (12660-012, Invitrogen), 20% Knockout Serum Replacement (10828-028, Invitrogen), 0.1 mM β-mercaptoethanol, 0.1 mM MEM Non-Essential Amino Acids (Invitrogen) and 10 ng/ml basic Fibroblast Growth Factor (Miltenyi Biotec) on 0.05% Gelatin for 4 days. Media was changed every other day.
Alternatively, the four reprogramming factors were overexpressed using plasmid pCAG2LMKOSimO (Addgene, Plasmid 20866). The control empty plasmid was generated by removing the four gene-encoding ORF region. pCAG2LMKOSimO plasmid was first linearised by PvuI restriction enzyme (New England BioLabs) at site 12413. After linearisation, plasmid was purified with SureClean kit (Bioline). Transfection of SMCs with pCAG2LMKOSimO or control plasmid was performed using Basic SMC Nucleofector Kit (Lonza) as specified by manufacturer. Cells were then maintained in reprogramming condition same as described above for 4 days.