Illumina HiSeq 2500 sequencing; GSM2413717: SOM-1; Mus musculus; RNA-Seq

SOM-1

The classification of neurons into distinct types is an ongoing effort aimed at revealing and understanding the diversity of the components of the nervous system. Recently available methods allow us to determine the gene expression pattern of individual neurons in the mammalian cerebral cortex to generate powerful categorization schemes. For a thorough understanding of neuronal diversity such genetic categorization schemes need to be combined with traditional classification parameters like position, axonal projection or response properties to sensory stimulation. Here we describe a method to link the gene expression of individual neurons with their position, axonal projection or sensory response properties. Neurons are labeled in vivo based on their anatomical or functional properties and, using patch clamp pipettes, their RNA individually harvested in vitro for RNAseq. With this method we can determine the genetic expression pattern of functionally and anatomically identified individual neurons. Overall design: single cortical neurons were patch clamped and the RNA harvested; single neuron mRNA profiles were generated by deep sequencing

Correlating anatomy and function with gene expression in individual neurons by combining in vivo labeling, patch clamp and single cell RNA-seq

Submitted by Gene Expression Omnibus on 30-NOV-2017

Mice aged P35 or older were anaesthetized with ketamine and xylazine (100mg*kg-1 and 10mg*kg-1, respectively), perfused transcardially with cold sucrose solution (in mM: NaCl, 83; KCl, 2.5; MgSO4, 3.3; NaH2PO4, 1; NaHCO3, 26.2; D-glucose, 22; sucrose, 72; and CaCl2, 0.5, bubbled with 95% O2 and 5% CO2) and decapitated, and the visual cortex was cut into 300µm coronal or tangential sections in cold sucrose solution. Slices were incubated in sucrose solution in a submerged chamber at 34°C for 30min and then at room temperature (21°C) until used for RNA harvest from patch-clamped cells. RNA was harvested as described previously (Pfeffer et al., 2013). In brief, slices were mounted under a 40x 0.8 NA water lens and superfused with sucrose solution (same as in slice preparation). Cells were identified using GFP or tdTomato  fluorescence and visualized with IR-DIC. Cells were then patched with regular RNAse free patch pipettes (2-4 MOhm resistance) in whole-cell mode using the voltage-clamp configuration. After seal establishment and break-in the cell was held at -70mV and the cell content including cell nucleus was harvested using negative pressure and expelled in an Eppendorf tube and frozen at -70C. Intracellular solution was RNAse free: in mM: CsMeSO4, 115; NaCl, 4; HEPES, 10; Na3GTP, 0.3; MgATP, 4; EGTA, 0.3; BAPTA(4Cs), 10; adjusted to pH 7.4 with CsOH; mOsm 295. The procedures lasted approximately 2-3 minutes per cell (from targeting to tube freezing). Single cell RNA-seq was carried out following the Cel-seq protocol (Hashimshony et al., 2016; Hashimshony et al., 2012). RT and second strand synthesis were performed using Superscript III (Invitrogen) and second strand synthesis kit (Invitrogen), respectively, before IVT amplification (Maxiscript T7, Ambion). 96 samples (up to 94 cells and 2-8 control samples) were handled in parallel to construct one sequencing library. After library fragmentation (Mg-Acetat + heat) and 3’ adaptor attachment, 11-15 cycles of PCR were used for library amplification (final concentration > 1ng/microL, library peak fragment size ~350 nt). Libraries were sequenced using Illumina HiSeq 2500 and HiSeq 4000 at the UCSD genomics core (1st read 13 cycles for the barcode, 2nd read 50 cycles for the 3’ end of the transcript).

Correlating Anatomy and Function with Gene Expression in Individual Neurons by Combining <i>in Vivo</i> Labeling, Patch Clamp, and Single Cell RNA-seq.

Correlating anatomy and function with gene expression in individual neurons by combining in vivo labeling, patch clamp and single cell RNA-seq

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