Ecori noti

The P8 gene codon that was optimized for expression in mammalian cells was synthesized by Cosmogenetech, Inc. The p8 DNA fragment (236 bp) was digested with EcoRI/NotI and cloned into the pCI-neo vector via the EcoRI/NotI site (Promega, Madison, WI) (Table 1). The construct was then transformed into E. coli DH5α for amplification. All restriction enzymes were purchased from New England BioLabs (Ipswich, MA).
Colorectal cancer (DLD-1) cells were transfected with plasmid DNA (pCI-neo and pCI-neo-p8). The day before transfection, DLD-1 cells were plated in 6-well plates at a density of 7 × 10⁵ cells per well. After incubating overnight, cells were transfected using Lipofectamine 3000 (Invitrogen) in accordance with the manufacturer’s instructions [31 (link)]. The transfected cells were selected in RPMI 1640 containing antibiotics. (G-418) (Sigma, St. Louis, MO, USA).

The protein-coding
region
of human OR5K1 (NM_001004736.3) was derived from our previously published
OR library.^{99 (link)} Amplification was carried
out in a touchdown approach using gene-specific primers (Table S2): an initial denaturation (98 °C,
3 min) and ten cycles consisting of denaturation (98 °C, 30 s),
annealing (60 °C, decreasing 1 °C per cycle down to 50 °C,
30 s), and extension (72 °C, 1 min), followed by 25 cycles of
denaturation (98 °C, 30 s), annealing (50 °C, 30 s), and
extension (72 °C, 1 min), finishing with a final extension step
in the end (72 °C, 7 min). Insertion of nucleotides into expression
vectors was done with T4-DNA ligase (#M1804, Promega, Madison, USA)
via EcoRI/NotI (#R6017/#R6435, Promega, Madison,
USA) into the expression plasmid pFN210A^{100 (link)} and verified by Sanger sequencing using internal primers (Table S3) (Eurofins Genomics, Ebersberg, Germany).

The protein-coding region of human OR10A6 and OR2W1 (for accession numbers see Table 2) was amplified from human genomic DNA by polymerase chain reaction (PCR), using gene-specific primers (Table S2), ligated with T4-DNA ligase (#M1804, Promega, Madison, USA) either MfeI/NotI (#R3589S/ # R0189S, New England Biolabs, Ipswich, UK) or EcoRI/NotI (#R6017/ #R6435, Promega, Madison, USA) into the expression plasmid (#pFN210A SS-HaloTag® CMV-neo Flexi®-Vector, Promega, Madison, USA), and verified by Sanger sequencing (Eurofins Genomics, Ebersberg, Germany) using vector internal primers (Table S3).