Spin column

RNA was isolated from cell lines and human pancreatic islets via Trizol. Briefly, 300 µl of Trizol (Zymo Research, Irvine, CA) was added to each well. After homogenizing, the Trizol reagent was transferred to an Eppendorf tube containing 300 ul of 95% ethanol and thoroughly mixed. The mixture was transferred to a spin column (Zymo Research, Irvine, CA) and RNA isolation was performed following the manufacturer’s protocol. DNA was removed by treatment with DNase (QIAGEN, Germantown, MD) according the manufacturer’s protocol. RNA quality was monitored on 1% agarose gels, and RNA quantification was performed using Thermo Scientific Nanodrop One Microvolume UV-vis spectrophotometer (Thermo Fisher Scientific, Grand Island, NY). qRT-PCR was performed as described (19 (link)). Primer sequences used to determine relative transcript abundance are listed in Table S1.

All strains of animals were purchased from Jackson laboratory. Mice are maintained in Lab Animal Services Facility at Stowers Institute with a 14:10 light cycle, and provided with food and water ad libitum. Experimental protocols were approved by the Institutional Animal Care and Use Committee at Stowers Institute and in compliance with the NIH Guide for Care and Use of Animals. Total RNA was isolated from VNO epithelia of individual mouse using TRIzol solution (Thermo Fisher Scientific) followed by spin-column (Zymo Research) purification. Ribodepletion was performed using Ribo-Zero Gold rRNA Removal kit (Illumina) to remove rRNA from the sample prior to library preparation. Sequencing libraries were generated using TruSeq Stranded Total RNA Kit (Illumina) and sequenced as 125 bp paired-end stranded reads on Illumina Hi-Seq 2500 platform. Preliminary analysis including basecalling was performed using HiSeq Control Software (v2.2.58) with fastq files generated using bcl2fastq. FastQC [64 ] reports were generated for each sample to ensure sequencing quality. Trim Galore was used with default parameters to trim reads with leftover adapter sequence and low quality scores [65 ].

Glucose measurement was carried out using Glucose Assay kit (Sigma GA2020). Hemolymph from 30 flies aged 5 days in normal fly food was collected using Zymo Spin columns by pricking the thorax with a fine needle in ice-cold conditions by centrifugation at 15,000 rpm for 15 minutes. 1μl of hemolymph was added to the Glucose Assay Reagent and incubated at 37°C for 30 minutes. The reaction was stopped by adding 6M H2SO4. Absorbance was measured at 540 nm using a F200 TECAN 96 well micro-plate reader. Independent biological replicates of this experiment were performed; the number of replicates is available in the figure legends.

All buffers and NTPs were purchased from commercial sources. Dimethyl sulfate was purchased from Sigma Aldrich (77-78-1). Potassium borohydride was purchased from Santa Cruz Biotechnology (SC-250747). SuperScript II reverse transcriptase was purchased from Invitrogen. Marathon RT was purchased from Kerafast. DFHBI-1T was purchased from Lucerna Technologes. N-methyl mesoporphyrin IX (NMM) was purchased from Santa Cruz Biotechnology. DNAse I was purchased from Sigma Aldrich. DNA constructs and primers were ordered from Integrated DNA Technologies (IDT) and Twist Bioscience. Spin columns were purchased from Zymo Research. AMPure XP beads were purchased from Beckman Coulter. Barcoded sequencing oligos were purchased from New England BioLabs. AmpliScribe T7 High Yield Transcription Kit were purchased from Biosearch Technologies.

Cells were grown until appropriate OD₆₀₀, and aliquots were collected and treated with RNAprotect Bacteria Reagent (Qiagen). After 5 min, cells were harvested and resuspended in lysis buffer (RNase-free TE buffer, 10 mg ml⁻¹ lysozyme, 100 µg ml⁻¹). Trizol LS (Thermo Scientific) was used according to the manufacturer’s protocol to extract the total RNA. Samples were treated with DNase (Invitrogen) and purified using spin columns (Zymo Research). Superscript III reverse transcriptase (Invitrogen) was used to synthesize cDNA. qPCR reactions were amplified using Power SYBr Green PCR Master Mix (Applied Biosystems) with appropriate primer sets and the cDNA template.