GSM8183542: neurons, sorted, scRNA-seq; Mus musculus; RNA-Seq
For sample 1, we isolated single cells from the cNST as previously described with the following modifications. Briefly, mice were anaesthetized with isoflurane and transcardially perfused with ice-cold carbogenated (95% O2, 5% CO2) NMDG-HEPES-ACSF (93 mM NMDG, 2.5 mM KCl, 1.2 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 10 mM MgSO4, 1 mM CaCl2, 1 mM kynurenic-acid Na salt, 5 mM Na-ascorbate, 2 mM Thiourea, 3 mM Na-pyruvate, pH 7.4). The brainstems were rapidly extracted and sliced into 300 μm sections containing the cNST using a vibratome (Leica, #VT-1000S) in ice-cold NMDG-HEPES-ACSF solution with continuous carbogenation. The cNSTs were dissected, pooled (from 5 animals), and digested in Trehalose-HEPES-ACSF (92 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 2 mM MgSO4, 2 mM CaCl2, 1 mM kynurenic-acid Na salt, 2.5 wt/vol trehalose, pH 7.4) containing Papain (20 U ml-1, Worthington, #LK003150) and DNase I (25 U ml-1) at 35oC for approximately one hour. Using Pasteur pipettes with progressively narrowing tip diameters, the tissue was triturated in DNase I-containing (25 U ml-1) Trehalose-HEPES-ACSF solution to form single-cell suspension. The dissociated cells were passed through 40 μm filter and resuspended in Resuspension-ACSF (117 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 1 mM MgSO4, 2 mM CaCl2, 1 mM kynurenic-acid Na salt, 0.05% BSA, pH 7.4). The resulting cell suspension was processed by the Columbia Genome Core to encapsulate and barcode individual cells using the 10X Genomics Chromium system. For sample 2, we used TRAP2; Ai9 mice that were TRAP-labeled with tdTomato in response to LPS. Cells from the cNST were isolated as described above. The cell suspension was stained with DRAQ5 (Thermoscientific, #62254) and Calcein Violet (Thermoscientific, #C34858) to label viable cells, prior to FACS. A total of 288 tdTomato+ LPS TRAPped cells and 96 tdTomato- cells were sorted into 96-well plates pre-loaded with cell lysis buffer containing 0.1% TritonX-100, SuperaseIN (Ambion, #AM2694), 1 mM dNTP and 1 μM capture primer (i.e. bar-coding). cDNA was synthesized using Maxima Reverse Transcriptase (Thermoscientific, #EP0753) according to manufacturer's instruction. cDNA from all the wells/cells was combined, followed by clean-up using Silane beads (Thermoscientific #37002D). Pooled cDNA was amplified using Kapa HotStart Mix with SMART PCR primer (0.2 μM), and then purified using AMPureXP beads (Beckman Coulter Life Sciences #A63880). 0.6 ng of cDNA was used as input to prepare libraries using Nextera XT kit (Illumina #FC-131-1024).