scRNA-seq of mouse sympathetic ganglia cells under control, cold-exposure, and microbiota-depletion conditions
scRNA-seq of mouse sympathetic ganglia cells under control, cold-exposure, and microbiota-depletion conditions
Single-cell suspensions were prepared from 10-14 C57BL/6J mice (8-12 weeks old), with equal numbers of males and females. Animals were anesthetized and euthanized, followed by cardiac perfusion of ice-cold NMDG-ACSF (92 mM NMDG-Cl, 2.5 mM KCl, 1.25 mM NaH2PO4, 30 mM NaHCO3, 25 mM Glucose, 20 mM HEPES, 10 mM, MgSO4, 0.5 mM CaCl2, 2mM Thiourea, 5 mM Na-ascorbate, 3 mM Na-pyruvate and bubbled with 95% O2/5% CO2). The sympathetic ganglia were rapidly extracted under the dissecting microscope and placed in ice-cold NMDG-ACSF containing 30 µM Actinomycin and bubbled with 95% O2/5% CO2. The connective tissue surrounding the ganglion was carefully peeled and removed. The ganglia tissue were then transferred to 2 ml enzyme mix buffer containing 50 U/ml PAPL (LS003119, Worthington), 0.4 mg/ml Trypsin, 1.2 mg/ml Collagenase D, 1mg/ml Dispase, 10 mM Cysteine, 0.2 mg/ml Deoxyribonuclease I, 0.5 mM EDTA, dissolved in ACSF (124 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 24 mM NaHCO3, 12.5 mM Glucose, 5 mM HEPES, 1 mM, MgSO4, 2 mM CaCl2, and bubbled with 95% O2/5% CO2) containing 10 µM Actinomycin, and prewarmed to 36 °C. The ganglia tissue was cut into smaller pieces and digested in the enzyme mix buffer for 1 hour at 36 °C. After enzymatic digestion, the ganglia tissue was gently triturated into single-cell suspension with using fire-polished glass Pasteur pipettes with tip diameters of 600, 300 and 150 μm. The cell suspension was brought up to 4 ml and passed through a 40 μm cell strainer (43-10040, pluriStrainer). Cells were centrifuged at 300 g for 5 minutes at 4 °C. After discarding the supernatant, the cell pellet was washed with 2 ml ice-cold and 95% O2/5% CO2 bubbled ACSF.  After a second centrifugation, the cell pellet was resuspended in 40 μl ACSF, containing 0.05% BSA and kept on ice while the cell density was counted with a haemocytometer. The final cell suspension volume estimated to retrieve 20,000 single-cell transcriptomes was loaded to the Chromium GEM-X 3' chip (PN 2001097, 10x Genomics). The Chromium GEM-X Single Cell 3' Kit v4 (PN-1000686, 10x Genomics) and Dual Index Kit TT Set A (PN-1000215, 10x Genomics) were used. Chronic cold exposure Adult mice (8-12 weeks) were maintained under standard conditions. For chronic cold exposure, home cages were transferred daily to a pre-chilled 4 °C cold-room for 2 h at the same time slot during the light cycle on 7 consecutive days. During each exposure, food and water were removed. Animals were monitored for general health. The next day after the final session, mice were euthanized for tissue collection.  Gut microbiome depletion Adult mice of at least 8 weeks old were maintained on water containing broad-spectrum antibiotics in home cage for up to three weeks. First antibiotic cocktail consists of ampicillin (1 g/L), vancomycin (0.5 g/L), neomycin (1 g/L), gentamicin (0.1 g/L), and erythromycin (0.01 g/L). The second recipe contains ampicillin (1 g/L), vancomycin (0.5 g/L), neomycin (1 g/L), and metronidazole (0.5 g/L) supplemented with 10% sucrose. For the control group for the sweet taste, 10% sucrose water will be provided ad lib. Water with antibiotics were filtered with a 0.22-μm filter, and changed at least once per week.   Single-cell suspensions were processed using the Chromium Next GEM Single Cell 3′ Reagent Kits v4 (10x Genomics) according to the manufacturer's protocol. Briefly, individual cells were encapsulated together with barcoded gel beads and reverse transcription reagents into nanoliter-scale Gel Bead-in-Emulsions (GEMs) using the Chromium Controller. Within each GEM, cell lysis and reverse transcription occurred simultaneously, generating barcoded first-strand cDNA from polyadenylated mRNA molecules captured by oligo-dT primers. After reverse transcription, emulsions were broken and cDNA was purified using silane magnetic beads, followed by PCR amplification to generate sufficient material for library construction. Amplified cDNA was enzymatically fragmented, end-repaired, A-tailed, and ligated to sequencing adapters containing unique sample indices. The final libraries were quantified, quality-checked, and sequenced on an Illumina platform according to the 10x Genomics recommendations. No external RNA spike-in controls were used. Amplified cDNA was enzymatically fragmented, end-repaired, A-tailed, and ligated to sequencing adapters according to the Chromium Next GEM Single Cell 3′ v4 workflow. Adapter-ligated products were purified and subjected to sample index PCR amplification to incorporate Illumina-compatible P5 and P7 sequences and unique sample indices. The completed libraries were purified using SPRIselect magnetic beads, quantified by fluorometric or qPCR-based methods, and quality-assessed by fragment analysis. Sequencing-ready libraries were loaded onto an Illumina platform for paired-end sequencing following 10x Genomics recommendations.