Journal: Nucleic Acids Research
Article Title: Using high-throughput barcode sequencing to efficiently map connectomes
Figure Lengend Snippet: Optimization of SYNseq components in HEK cells. ( A ) The presynaptic components of the SYNseq system, consisting of CLIP-Nrx1B-1xnλ and a GFP encoding barcode RNA. ( B ) The postsynaptic components of SYNseq, consisting of SNAP-Nlg1AB-1xnλ and a mCherry encoding barcode RNA. ( C and D ) A clear membrane staining of synPRE-P and synPOST-P can be observed after staining ( C ) synPRE-P expressing HEK cells with CLIP-Surface488 and ( D ) synPOST-P expressing HEK cells with SNAP-Surface488. Scale bar = 5 μm. ( E ) Western blot analysis shows that synPRE-P and synPOST-P can be specifically crosslinked by addition of a small molecule BG-PEG-Biotin-PEG-BC crosslinker. A crosslinked product is only produced when both synPRE-P and synPOST-P were expressed in HEK cells and the crosslinker was added before lysis (lane 5). Arrow = crosslinked band; star = uncrosslinked synPRE-P or synPOST-P. ( F ) synPRE-P and synPOST-P specifically and strongly bind to their respective barcode mRNAs as evident in RNA-IPs from transiently transfected HEK cells, after membrane tagging with BG–PEG–biotin–PEG–BC. We show qRT-PCR analysis of three independent RNA-IP experiments and western blot analysis of a representative sample.
Article Snippet: Tagging with BG/BC derivatives We obtained all BG- and BC-functionalized derivatives, including the bifunctional cross-linkers (synthesis see above), CLIP-Surface488 and SNAP-Surface488 from New England Biolabs (NEB) and used them according to the manufacturer's instructions.
Techniques: Cross-linking Immunoprecipitation, Staining, Expressing, Western Blot, Produced, Lysis, Transfection, Quantitative RT-PCR