All constructs were created using standard molecular biology procedures and verified by Sanger sequencing. Detailed information about constructs for recombinant protein production and mammalian expression, recombinant protein sequences, and DNA oligonucleotides for construct generation can be found in Supplementary Tables 1 , 2 , and 3 , respectively. The ORFs of human VPS35L and VPS26C were previously described34 –36 . The mammalian expression vector for SNX17 was previously described37 . SNX31 mammalian expression vector was designed by GeneArt at Thermo Fisher Scientific. For insect cell expression of Retriever, human full-length VPS35L (untagged, synthesized as a codon-optimized GeneString from Thermo Fisher to improve expression), VPS26C (untagged), and VPS29 (isoform 2) containing a C-terminal (GGS)2-His6 tag were cloned in a modified pFastBac™ vector for insect cell expression as previously described34 . For bacterial expression of isolated VPS26C, codon-optimized GeneString (Thermo Fisher) was cloned in a pMalC2Tev vector34 . Bacterial expression vector of GST-SNX17 was previously described38 . Bacterial expression vectors of CCDC22 VBD and CCDC93 VBD were previously described34 . The CT 20 amino acids of SNX17, SNX31, LRMDA, TIMM23, PATE1, ARHGEF25, and HYOU1 were codon-optimized and cloned into a pGexTev vector using PCR.
Genestring
Manufactured by Thermo Fisher Scientific
The GeneString is a compact, high-performance laboratory instrument designed for nucleic acid analysis. It utilizes advanced sequencing technology to accurately analyze DNA and RNA samples. The GeneString provides reliable and precise data for various genomic research and diagnostic applications.
Automatically generated - may contain errors
5 protocols using genestring
1
Recombinant Protein Expression Protocols
2
Lentiviral Overexpression of SirT3 in Cardiomyocytes
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SirT3 cDNA was synthesized as gene string from ThermoFisher Scientific and cloned in pLV lentiviral backbone. Lentiviral overexpression of SirT3 in cardiomyocytes was mediated by transduction of lentivirus from pLV plasmid carrying cDNA sequence of SirT3 and empty control [65 (link)]. For lentiviral production, HEK293T was transfected with pLV plasmid together with helper plasmids (vsvg, gagpol, rev, and NovB2) using CaCl2. Medium was changed after 8 hours of transfection. Viral supernatant was collected 48 to 72 hours later [66 (link)]. Cardiomyocytes were transduced with lentivirus, and after 3 days, puromycin selection was initiated to remove nontransduced cells. After puromycin selection, overexpression was confirmed by western blot [67 (link)].
3
Overexpression of miR-998 and miR-29a
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HeLa cells were cultured in DMEM+10% FBS, and were transfected with the X-treme Gene HP transfection reagent (Roche) according to the manufacturer's protocol. Cells were harvested 24–48 hours post-transfection. pcDNA3/mir-998 generated by PCR amplification of the mir-998 gene and standard molecular cloning techniques. pcDNA3/hsa-mir-29a was generated by insertion of a GeneString (Invitrogen) with the mir-29a gene sequence in pcDNA3. Insert sequences were verified by sequencing analysis. Sequences are in Table S4 . Firefly and Renilla luciferase activity were measured using the Dual Luciferase Assay protocol (Promega).
4
Generating PARP13 and TRAILR4 constructs
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GFP-PARP13 has been described previously1 . To generate SBP-PARP13, GFP was substituted with streptavidin binding peptide tag using NheI and BspEI. PARP13 ΔZnF and PARP13 RNA binding point mutants were generated using GeneString (Invitrogen) flanked by XhoI/BstXI, which are internal sites in PARP13. PARP13ΔZnF features a deletion from nt228 to nt669.
The psiCHECK2 vector encoding Renilla and Firefly luciferase genes was purchased from Promega. TRAILR4 ORF was purchased from Origene (SC117708). A SalI site was introduced after the TRAILR4 stop codon using a Gene String flanked by PpuMI and ScaI, which are internal sites in TRAILR4 cDNA. The 3’UTR of TRAILR4 was then introduced downstream the Renilla luciferase in psiCHECK2 using SalI/XhoI and NotI digestion. psiCHECK2 + GAPDH 3’UTR is a kind gift from Dr. Benilde Jimenez. Truncations of TRAILR4 3’UTR were generated by PCR using primers with XhoI/NotI overhangs. psiCHECK2+TRAILR4 3’UTR was used as a template. Fragments were designed based on TRAILR4 3’UTR folding prediction (RNAFold) so as to preserve high-probability folding structures34 (link).
The psiCHECK2 vector encoding Renilla and Firefly luciferase genes was purchased from Promega. TRAILR4 ORF was purchased from Origene (SC117708). A SalI site was introduced after the TRAILR4 stop codon using a Gene String flanked by PpuMI and ScaI, which are internal sites in TRAILR4 cDNA. The 3’UTR of TRAILR4 was then introduced downstream the Renilla luciferase in psiCHECK2 using SalI/XhoI and NotI digestion. psiCHECK2 + GAPDH 3’UTR is a kind gift from Dr. Benilde Jimenez. Truncations of TRAILR4 3’UTR were generated by PCR using primers with XhoI/NotI overhangs. psiCHECK2+TRAILR4 3’UTR was used as a template. Fragments were designed based on TRAILR4 3’UTR folding prediction (RNAFold) so as to preserve high-probability folding structures34 (link).
5
Generating PARP13 and TRAILR4 constructs
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