Journal: bioRxiv
Article Title: Deep mutational scan of the pore of the cold-sensing TRPM8 channel
doi: 10.64898/2026.04.28.721489
Figure Lengend Snippet: (A) Cartoon representation of the experimental strategy used in this study. Libraries were generated from a pooled oligo library containing fragments coding for each type of single-residue missense variant within positions 927-974 of the rat TRPM8 channel. Oligo fragments were PCR-amplified and cloned into ‘acceptor’ expression plasmids by Golden Gate cloning using BsmBI sites . (B) The plasmid library was expressed in iCasp9 cells , and cells expressing mCherry were sorted by flow cytometry into groups of low, medium, or high GCaMP6s fluorescence intensity after stimulation with cold or 2 mM menthol. The genomic DNA was extracted from the sorted cells, from which the TRPM8 coding region was amplified by PCR, cleaved by a restriction endonuclease to reduce amplicon size, and prepared for next-generation sequencing. (C) NGS read counts per variant in the plasmid library, showing homogenous variant representation. (D) mCherry fluorescence intensity distribution in the library cells that were sorted. (E) GCaMP6s fluorescence intensity distribution of the library cells, measured at room temperature (dashed black curves, not sorted), or after stimulation with cold (blue) or 2 mM menthol (green) during cell sorting. Dashed red lines denote the three cell-sorting bins. (F) Deep mutational scanning score distribution for cold and menthol experiments. (G) Correlation between individual variant scores from two biological replicates. (H) GCaMP6s fluorescence responses in four silent mutations included in the library (gray traces) relative to cells expressing WT.
Article Snippet: To generate the acceptor plasmid for the library, we used restriction sites MfeI and SphI, which flank the sequence coding for the transmembrane domain of TRPM8, to clone a double stranded DNA insert (Twist Bioscience) that included BsmBI sites flanking the variable-sequence region of the library from positions 927 to 974.
Techniques: Generated, Residue, Variant Assay, Amplification, Clone Assay, Expressing, Cloning, Plasmid Preparation, Flow Cytometry, Fluorescence, Next-Generation Sequencing, FACS
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