HEK293T (ATCC) were grown in DMEM, 10% fetal calf serum, and 1% penicillin/streptomycin at 37°C, 5% CO 2 and transfected using Lipofectamine 2000 (Invitrogen/Life Technologies) according to manufacturer’s protocol. 1 μg of human Adrα2A or TRPC4β was transfected for independent expression with 0.2 μg GFP. For coexpression experiments, equal concentrations of Adrα2A and TRPC1, TRPC3, TRPC4, or TRPC6 were used. Constitutively active (Q205L) or dominant-negative (G203T) Gα i2 mutants were included in the transfection mix for some experiments. For Olfr558 experiments, 1.5 μg of Olfr558 was transfected with several constructs used enhance expression and signaling of olfactory receptors in heterologous systems: 1 μg of receptor-transporting protein 1 short (RTP1S), guanine nucleotide exchange factor B (Ric8b), and Gα olf/15 , and 0.2 μg GFP for cellular identification. Mock transfection experiments were performed by transducing all constructs except Olfr558. Human Adrα2A was from Genscript (Piscataway, NJ), Gα i2 mutants from cDNA Resource Center; TRPC1, TRPC3, and TRPC6 were cloned into pcDNA3 in the Julius lab; TRPC4 was a gift from J. Jeon and M. Zhu; Olfr558 constructs were gifts from H. Matsunami (Addgene) and were tagged with the first 20 residues of human rhodopsin to increase expression; RTP1S, Ric8b, and Gα olf/15 were gifts from A. Chang. lentiCRISPR v2 was a gift from F. Zhang (Addgene).

Retrogradely traced colonic sensory neurons neurons were isolated from adult male mice following injection of cholera toxin subunit B conjugated to AlexaFluor 488 (CTB-488; Invitrogen, Carlsbad, CA) at three sites sub-serosally within the wall of the distal colon. After 4 days, lumbosacral (LS) dorsal root ganglion neurons were isolated and cultured as previously described (). Briefly, mice were euthanized by COinhalation and lumbosacral dorsal root ganglia (DRGs) (L6-S1) from retrogradely traced mice were surgically removed and were digested with 4 mg/mL collagenase II (GIBCO, Life Technologies) plus 4 mg/mL dispase (GIBCO) for 30 min at 37°C, followed by 4 mg/mL collagenase II for 10 min at 37°C. Neurons were then mechanically dissociated into a single-cell suspension via trituration through fire-polished Pasteur pipettes. Neurons were resuspended in DMEM (GIBCO) containing 10% FCS (Invitrogen), 2mM L-glutamine (GIBCO), 100 μM MEM non-essential amino acids (GIBCO), 100 mg/ml penicillin/streptomycin (Invitrogen) and 100ng/ml NGF (Sigma). Neurons were spot-plated on coverslips coated with poly-D-lysine (800 μg/ml) and laminin (20 μg/ml) and maintained at 37°C in 5% CO

Adult male ChgA-GFP mice aged 6-10 weeks were used to generate intestinal organoids, as previously reported (). Briefly, the small intestine was isolated and washed with cold PBS and crypts were isolated following dissociation in EDTA. Isolated crypts were suspended in Matrigel. Following polymerization, organoid growth media containing murine epidermal growth factor (Peprotech), noggin (Peprotech), and 10% R-spondin conditioned media was added and refreshed every 3-4 days. Organoids were maintained at 37°C, 5% COand propagated weekly. For Caimaging, Matrigel was removed from organoids, they were loaded with Fura-2AM, fenestrated by mechanical disturbance, and then immediately placed in the imaging chamber containing Cell-Tak (Corning)-coated coverslips. For electrophysiology, organoids were mechanically dissociated and placed on Cell-Tak coated coverslips in the recording chamber.

Mouse breeding, housing, and use was approved by the UCSF Animal Care and Use or University of Adelaide and Flinders University Animal Ethics Committees. Adult male mice of C57BL/6 background (Jackson Labs) aged 12-16 weeks with an average weight of ∼29 g were used for ex vivo afferent nerve recordings and colonic sensory neuron imaging. Male mice were used in all studies to account for effects from sex or genetic background. Animals were housed in groups (2-5 mice/cage) in a specific and opportunistic pathogen free facility, fed Jackson lab diet (5K52 JL RAT & MOUSE/AUTO 6F), provided with environmental enrichment (shelter, nesting material, etc.), and had normal immune status. Reporter mice were gifts from T. Schwartz (ChgA-GFP) and M. Scanziani (5HT 3 R-GFP).

Method Details

CRISPR-mediated gene disruption Sanjana et al., 2014 Sanjana N.E.

Shalem O.

Zhang F. Improved vectors and genome-wide libraries for CRISPR screening. 2+ responses compared with wild-type organoids, so were grouped with other controls in some analyses. gRNA sequences were designed with the Cas9 design target tool ( http://crispr.mit.edu ) and inserted into the Cas9-containing lentiCRISPR v2 vector (). Primers used to design the specific gRNA target were: Olfr558 forward (5′ to 3′) CACCGagcacagtggcatgccgtag; Olfr558 reverse (5′ to 3′) AAACctacggcatgccactgtgctC. Lentivirus was produced by transfecting HEK293T cells with psPAX2, pVSVG and LentiCRISPR v2 with Olfr558 gRNA using Fugene HD (Roche) according to the manufacturer’s instructions. Virus was concentrated and re-suspended in organoid growth medium. As a control for both sequencing and functional experiments, organoids were infected with empty Cas9-containing LentiCRISPR v2 vector. Vector-infected organoids expressed wild-type Olfr558 sequence and exhibited similar isovalerate-induced Caresponses compared with wild-type organoids, so were grouped with other controls in some analyses. Two days before infection, intestinal organoids were grown in a 24-well culture plate with growth medium supplemented with 5 μM CHIR99021 (Sigma) and 10 mM nicotinamide (Sigma) to increase stem cell population. Stem cell-enriched organoids were broken down into single-cells, viral mix was added, and cells were transferred to a 48-well plate that was centrifuged at 600 g for 60 min (spinoculation) and placed in the incubator for another 6 hr at 37°C. Cells were collected, re-suspended in Matrigel, and transferred into a 24-well culture plate. After two days of recovery, selection was carried out using puromycin (6 μg/ml) for three days. After selection, growth medium was supplemented with 5 μM CHIR99021 and 10 mM nicotinamide. Organoids were then treated with TrypLE (Life Technologies) at 37°C for 5 min to achieve single cells which were plated onto 96-well culture plates for clonal selection. Growth medium was supplemented with 5 μM CHIR99021, 10 μM Y-27632 (Sigma) and 10 mM nicotinamide during the first two days after plating to enrich for stem cells and prevent apoptosis. The medium was then changed to growth medium supplemented with 5 μM CHIR99021 and 10 mM nicotinamide for another three days and then normal growth medium afterward. Single organoids were then collected and used for clonal expansion. To verify clonal populations genetic disruption, genomic DNA was isolated using QuickExtract DNA extraction solution (Epicenter) and PCR amplified using Phusion polymerase (NEB) and the following primers for Olfr558: F: ctttgcatgctttctggcct; R: tgcaggtgttcttccattcca. Products were cloned into Topo vectors (Agilent) and sequenced.

Electrophysiology Recordings were carried out at room temperature using a MultiClamp 700B amplifier (Axon Instruments) and digitized using a Digidata 1322A (Axon Instruments) interface and pClamp software (Axon Instruments). Data were filtered at 1 kHz and sampled at 10 kHz and leak-subtracted online using a P/4 protocol for voltage step protocols. Membrane potentials were corrected for liquid junction potentials. EC cells were identified by GFP expression and recordings were made using borosilicate glass pipettes polished to 7 - 9 MΩ. Recording pipettes used for HEK293 were 3 - 4MΩ. Unless stated otherwise, a standard Ringer’s extracellular solution for EC and HEK293 cell experiments contained (mM): 140 NaCl, 5 KCl, 2 CaCl 2 , 2 MgCl 2 , 10 HEPES, 10 glucose, pH 7.4. Intracellular solution for recording K+ currents from EC cells contained: 140 K-gluc, 5 KCl, 1 MgCl 2 , 10 K-EGTA, 10 HEPES, 10 sucrose, pH 7.2. Intracellular solution for current-clamp recordings contained: 140 K-gluc, 5 NaCl, 1 MgCl 2 , 0.02 K-EGTA, 10 HEPES, 10 sucrose, pH 7.2. Other EC cell and 5HT 3 R recordings used the following intracellular solution: 140 CsMeSO 4 , 5 NaCl, 1 MgCl2, 10 Cs-EGTA, 10 HEPES, 10 sucrose, pH 7.2. Intracellular solution for Adrα2A-expressing cells contained 0.1 Cs-EGTA. For EC recordings, holding potential was −90 mV and currents were elicited by 500ms ramps from −100 mV to +100 mV or 200ms steps in 10 mV increments. G-V relationships were derived from I-V curves by calculating G: G = I Ca / (V m -E rev ) and were then fit with a Boltzman equation. Voltage-dependent inactivation was measured during −10 mV voltage pulses following a series of 1 s prepulses ranging from −110 to 60 mV in 10 mV increments. Voltage-dependent inactivation was quantified as I / I max , with I max occurring at the voltage pulse following a −110 mV prepulse. Adrα2A-associated experiments were carried out using a protocol that consisted of 10 s holding voltage at −60mV followed by a 500 ms ramp from −100 mV to +100 mV that returned to −60 for an additional 10 s, and this protocol was repeated consecutively for ∼10 min or more. For 5HT 3 R biosensor recordings, whole-cell configuration was achieved and cells were lifted from coverslips and moved immediately adjacent to GFP-labeled EC cells. Voltage has held constant at −80 mV as solutions were washed on and off with local perfusion. Responses were normalized to peak current induced by mCPBG.

Calcium imaging EC and HEK were loaded with 10 μM Fura-2-AM (Invitrogen) and 0.01% Pluronic F-127 (wt/vol, Invitrogen) for 1 h in Ringer’s solution. 340 nm to 380 nm ratio was acquired using MetaFluor software. EC cells were identified by GFP expression and responses were normalized to increased fluorescence ratio elicited by high extracellular K+ (K+, 140 mM) at the end of the experiment. In most experiments, only one EC cell was identified in the field of view, thus we quantified data from single cells. In somewhat rare cases when two EC cells were observed in the same field of view, responses were averaged. Dorsal root ganglion neurons were cultured for 24 hr, incubated with 2.5 μM Fura2-AM and 0.02% (v/v) pluronic acid for 30 min at room temperature in modified Ringer’s solution containing (mM): 145 NaCl, 5 KCl, 1.25 CaCl 2 , 1 MgCl 2 , 10 glucose, 10 HEPES. After a brief wash, coverslips were transferred to the recording chamber and Ca2+ responses were measured at room temperature. Colonic DRG neurons were identified by the presence of the 488 tracer and viability was verified by responses to 25 mM KCl. All pharmacological agents were delivered by local perfusion with exception of 1 μM U73122, 100 μM gallein, 200 ng/ml cholera toxin, or 200 ng/ml pertussis toxin, 10 μM SQ22536, which were preincubated. Associated vehicle control experiments were performed. In experiments using HEK293, construct-expressing cells identified by GFP expression were quantified and responses were normalized to maximal responses elicited by 1 μM ionomycin at the end of the experiment. Concentrations and abbreviations of molecules used in Ca2+ imaging screening (in μM): 1 Capsaicin, 500 allyl isothiocyanate (AITC), 50 1-(m-chlorophenyl)-biguanide (mCPBG), 1 icilin, 200 N-butyryl-L-Homoserine lactone (C4-HSL), 200 N-hexanoyl-L-Homoserine lactone (C6-HSL), 200 N-3-oxo-dodecanoyl-L-Homoserine lactone (3OC12-HSL), 1 or 10 N-Formylmethionine-leucyl-phenylalanine (fMFL), 50 μg/ml lipopolysaccharide (LPS) from E. Coli, 500 indole, 500 sodium propionate, 500 sodium acetate, 500 sodium butyrate, 500 isobutyrate, 500 isovalerate, 500 sodium deoxycholate, 1 substance P, 100 histamine, 1000 glutamate, 100 tryptamine, 100 serotonin, 100 glycine, 100 gamma-aminobutyric acid (GABA), 100 dopamine, 100 epinephrine, 100 norepinephrine. Unless stated otherwise, concentrations of other pharmacological agents (in μM): 0.5 tetrodotoxin (TTX), 10 nifedipine, 0.3 ω-agatoxin IVA, 0.3 ω-conotoxin, 5 mibefradil, 1 epinephrine, 1 norepinephrine, 5 yohimbine, 10 isoproterenol, 5 prazosin, 5 clonidine, 5 propranolol, 10 phenylephrine, 5 U73122, 100 gallein, 200ng/ml pertussis toxin (PTX), 200ng/ml cholera toxin (CTX), 50 2-aminoethoxydiphenyl borate (2-APB), 10 ML204. Most drugs were from Tocris, HSLs and 4-hydroxynonenal were from Cayman Chemical, volatile fatty acids were from Sigma.

Transcriptome sequencing and analysis Intestinal epithelial cells from organoids were dissociated and immediately sorted by fluorescence-activated cell sorting (FACS) by the Laboratory for Cell Analysis at UCSF. ∼1% of total epithelial cells were GFP+ and collected. The remaining GFP- cells were retained for comparison. RNA from GFP+ and GFP- subgroups was then extracted and prepared for cDNA library generations using the SMARTer Ultra Low Input RNA kit followed by the Low Input Library Prep Kit (version 2, Clontech Laboratories, Inc.). cDNA quality was assessed via bioanalyzer using the High Sensitivity DNA kit (Agilent Technologies), and high quality samples were preserved for sequencing. PolyA cDNA libraries were sequenced on the Illumina Hi-Seq 4000 platform (QB3 Vincent J. Coates Genomic Sequencing Library), generating 150 bp paired-end reads. More than 100M reads were obtained. The quality of raw sequence reads was analyzed via FASTQC. Adapters were trimmed using Scythe, and sequence read ends were trimmed using Sickle. Reads were then aligned to the annotated mouse reference genome (mm10) using TopHat2 (version 0.7). Transcripts were assembled and relative abundance was estimated using Cufflinks and Cuffdiff tools. Gene ontology-based (GO) analyses were carried out using DAVID (version 6.8) to categorize the top ∼1000 transcripts annotated with ENSEMBL gene IDs that showed the greatest fold change between GFP+ and GFP- samples. The “biological process” set of GO terms was used in functional annotation of the enriched transcripts in the GFP+ sample over the GFP- sample, which was set as the background.

Histology Ishii et al., 2004 Ishii T.

Omura M.

Mombaerts P. Protocols for two- and three-color fluorescent RNA in situ hybridization of the main and accessory olfactory epithelia in mouse. Immunofluorescence (IF) was performed using 5 μm cryosections. Blocking was performed with 10% normal serum corresponding to secondary antibody species in 0.1% Triton-X and PBS at room temperature for 60 min. Primary antibodies were incubated overnight at 4°C at the indicated dilutions. Antibodies used were against ChgA (1:200, Santa Cruz), serotonin (1:10,000, Immunostar), Adrα2A (1:200, Affinity Bioreagents), tyrosine hydroxylase (1:500, Millipore), Synapsin (1:500, from R. Edwards), PSD-95 (1:200, Neuromab), Lysozyme (1:200, Dako), GLP-1 (1:200, Abcam), Substance P (1:1000, Penninsula). Alexa Fluor-conjugated secondary antibodies were used at 1:300-1000 (Millipore). In situ hybridization histochemistry was performed using digoxigenin- and fluorescein-labeled cRNA for mouse TRPC4 or Olfr558. Probes were generated by T7/T3 in vitro transcription reactions using a 500-nucleotide fragment of TRPC4 (nucleotides 1553 to 2053), and a 500-nucleotide fragment of Olfr558 cDNA (nucleotides 1000 to 1500). Hybridization was developed using anti-digoxigenin and anti-fluorescein Fab fragments, followed by incubation with FastRed and streptavidin-conjugated Dylight 488 according to published methods (). Epifluorescence imaging was performed on an Olympus IX51 microscope equipped with a DP71 imager and Nikon Eclipse Ti with a DS-Qi2 imager. Confocal imaging was performed on Nikon Ti microscope with Yokogawa CSU-22 spinning disk. Images were assembled in Photoshop and ImageJ. Surface rendering was performed using UCSF Chimera.