Journal: Nature Communications
Article Title: rec-YnH enables simultaneous many-by-many detection of direct protein–protein and protein–RNA interactions
doi: 10.1038/s41467-018-06128-x
Figure Lengend Snippet: rec-YnH workflow. (I) ORF-coding or RNA-coding fragments are transferred into rec-Y2H or rec-Y3H screening vectors by batch Gateway or Gibson reactions (see Supplementary Figs. , ). This step is done only once; the resulting libraries are used for several full-screen replicates. (II) pBWH and pAWH libraries (bait and prey rec-Y2H screening libraries) or pMS22H and pAWH libraries (RNA and prey rec-Y3H screening libraries) are linearised by homing enzyme digests and co-transformed into yeast in batch. The transformed yeast pool is split in a recombination–selection (RS) medium (–Trp, omitted in the figure for simplicity; see Supplementary Fig. for details) and a recombination–interaction–selection (RIS) medium (–Trp and different selection markers available, see Supplementary Table ). rec-YnH screening is done in liquid–gel cultures. Only correctly fused vectors contain a yeast origin of replication and a TRP1 marker, and are thus able to grow in RS or RIS media. (III) Cells are harvested by centrifugation, the fused plasmid libraries are isolated, and the DNA is fragmented by Covaris and re-circularised by intramolecular ligation. In a first PCR reaction, circular fragments containing the 3′ ends of both a bait and a prey are specifically amplified from the pool of fragments, adding R1/R2 Illumina adaptors. A second PCR step then adds a multiplexing index and P5/P7 Illumina attachment sequences, thereby creating a library of fused bait-coding and prey-coding sequences with conserved C-termini (see Supplementary Fig. for details). (IV) Paired-end sequencing is used to readout library complexity and clone representation (RS condition) and bait–prey interactions (RIS condition). An analysis pipeline normalises the obtained reads, corrects for sequencing depth artefacts, and overrepresented clones (see Fig. and Supplementary Fig. )
Article Snippet: To minimise PCR bias, circular DNA was split into 10 independent PCR reactions, with Q5 High-Fidelity 2X Master Mix (New England Biolabs), and primers Pr4seq_F_TS_R1 (5′-CCCTACACGACGCTCTTCCGATCTCGCTGCAGGTCGACGGATC-3′) and Pr4seq_R_TS_R2 (5′-TTCAGACGTGTGCTCTTCCGATCTGCAGCTCGAGCTCGATGGATC-3′).
Techniques: Transformation Assay, Selection, Marker, Centrifugation, Plasmid Preparation, Isolation, Ligation, Amplification, Multiplexing, Sequencing, Clone Assay
