BsiWI-HF®

Protocol tips

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	Subsequently, lenti-dCas9-linkerGGGGS13-BioID2A-Blast was digested with BsiWI-HF and BamHI-HF. PCR amplified MTA2 sequence and backbone from BsiWI-HF/BamHI-HF digestion of lenti-dCas9-linkerGGGGS13-BioID2A-Blast were subjected to Gibson Assembly.

Protocol tips
Subsequently, lenti-dCas9-linkerGGGGS13-BioID2A-Blast was digested with BsiWI-HF and BamHI-HF. PCR amplified MTA2 sequence and backbone from BsiWI-HF/BamHI-HF digestion of lenti-dCas9-linkerGGGGS13-BioID2A-Blast were subjected to Gibson Assembly.

Publication protocol

"Humanized BioID2 sequence together with GGGGS13 linker was amplified from Addgene plasmid ID no. 74224 (ref. 65) by introducing the BsrG1 and BamHI cloning sites. The fragment was cloned in lentiCas9-Blast backbone (Addgene plasmid no. 52962) carrying dCas9 (dCas9 sequence from Addgene plasmid no. 47948) sequence between BamHI and BsiWI cloning sites. Together plasmid was initially reconstructed as lenti-dCas9-linkerGGGGS13-BioID2A-Blast with BioID2-linker and dCas9 sequence as one ORF. MTA2 sequence was obtained from Harvard Plasmid Data Base (ID no. HsCD00337978) and PCR amplified using following primers with complementary overhangs (see primer sequences in Supplementary Table 3). Subsequently, lenti-dCas9-linkerGGGGS13-BioID2A-Blast was digested with BsiWI-HF and BamHI-HF. PCR amplified MTA2 sequence and backbone from BsiWI-HF/BamHI-HF digestion of lenti-dCas9-linkerGGGGS13-BioID2A-Blast were subjected to Gibson Assembly. In parallel, the oligos encoding Kozak, start codon, FLAG and NLS (SV40) sequences and BsiWI/BamHI compatible ends (Supplementary Table 3) were annealed and ligated into BamHI/BsiWI digested lenti-dCas9-linkerGGGGS13-BioID2A-Blast vector to produce a control plasmid.

To produce lentivirus HEK293T cells were transfected with 13.3 μg of psPAX2, 6.7 μg VSV-G and 20 μg of the lentiviral construct plasmid (MTA2-BioID2/NLS-BioID2) using 180 μg of linear polyethylenimine. Lentiviral supernatant was collected after 72 h post-transfection and subsequently concentrated by ultracentrifugation. MTA2 biallelic deleted HUDEP-2 cells (MTA2–/–) were transduced with 5 μl of concentrated lentivirus. Following 24 h of transduction, cells were exposed to 10 μg ml–1 blasticidin in HUDEP-2 expansion media. Blasticidin resistant colonies were expanded and tested for MTA2-BioID2 expression and function using immunoblot and intracellular HbF staining, respectively."

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