The human eighth FLG repeat unit (324 amino acids) was selected for this study (Figure 1A ). The WT nucleotide sequence was optimized for E. coli codon usage bias (strain K12) using a codon optimization tool (Integrated DNA Technologies) (Supplementary Figure 1 ). The WT and E. coli codon-optimized FLG coding sequences (so-called “Codon Opt” further on) were synthesized (Integrated DNA Technologies) and cloned into plasmids pJL1 and pETBlue-1 by Gibson Assembly (Gibson et al., 2009 (link)) for CFPS and in vivo protein synthesis, respectively. 6xhistidine tag was added to the C-terminal end of FLG during PCR. E. coli DH5α competent cells were used for the cloning host. The sequences have confirmed by DNA Sanger-Sequencing using the 3130xl Genetic Analyzer (Applied Biosystems). The recombinant plasmids were isolated by plasmid maxiprep kit (Invitrogen, Waltham, MA). A schematic cloning workflow is described in the Supplementary Figure 2 .
Plasmid maxiprep kit
Manufactured by Thermo Fisher Scientific
The Plasmid maxiprep kit is a laboratory tool used for the large-scale purification of plasmid DNA. It provides a reliable and efficient method to isolate high-quality plasmid DNA from bacterial cultures.
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4 protocols using plasmid maxiprep kit
1
Optimized Bacterial Expression of Human FLG
2
Porcine BAC Probe Preparation for FISH
3
Pro-neural Gene Overexpression
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The pro-neural genes Ascl1, Neurog2, or Sox2 were expressed under control of an internal chicken β-actin promoter with cytomegalovirus enhancer (pCAG) together with DsRed or GFP behind an internal ribosomal entry site (pCAG-Ascl1-IRES-DsRed, pCAG-Neurog2-IRES-DsRed, and pCAG-Sox2-IRES-GFP). For control experiments, cultures were transfected with plasmids encoding only DsRed or GFP (pCAG-IRES-DsRed or pCAG-IRES-GFP) (Heinrich et al., 2010 (link); Karow et al., 2012 (link)). Plasmid stocks were prepared in Escherichia coli and purified using the endotoxin-free Maxiprep plasmid kit (Invitrogen). DNA concentration was adjusted to 1 μg/μL in TE buffer endotoxin free, and plasmids were stored at −20°C.
4
Plasmid Construction for Neural Induction
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Plasmids contain the internal chicken β-actin promoter fused with a cytomegalovirus enhancer (pCAG), the coding sequence for either Ascl1 or Neurog2, an internal ribosomal entry site (I) and coding sequences for either DsRed or GFP (pCAG-Ascl1-I-DsRed, pCAG-Neurog2-I-DsRed and pCAG-Neurog2-I-GFP). Control plasmids encode only DsRed or GFP (pCAG-I-DsRed or pCAG-I-GFP).
Plasmid stocks were prepared in Escherichia coli and purified using an endotoxin-free Maxiprep plasmid kit (Invitrogen). DNA concentration was adjusted to 1 μg/μL in endotoxin free TE buffer, and plasmids were stored at −20°C.
Plasmid stocks were prepared in Escherichia coli and purified using an endotoxin-free Maxiprep plasmid kit (Invitrogen). DNA concentration was adjusted to 1 μg/μL in endotoxin free TE buffer, and plasmids were stored at −20°C.
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