NextSeq 500 sequencing; GSM2085880: GAD_posterior_cell_1; Mus musculus; RNA-Seq

GAD_posterior_cell_1

The preoptic area (POA) of the hypothalamus is known to be crucial for sleep generation, but the spatial intermingling of sleep- and wake-promoting neurons makes it difficult to dissect the sleep control circuit. Here we identified a population of POA sleep-promoting neurons based on their projection target. Using a lentivirus for retrograde labeling with channelrhodopsin-2 (ChR2) followed by optogenetic manipulation and recording, we found that the POA GABAergic neurons projecting to the tuberomammillary nucleus (TMN) are both sleep active and sleep promoting. Cell type- and projection-specific rabies tracing revealed the presynaptic inputs to these neurons, including an amygdala GABAergic input that promotes wakefulness. Using single-cell RNA-seq, we identified several molecular markers for these neurons, and optogenetic activation of the POA neurons labeled by these markers confirmed their sleep-promoting effects. Together, these findings define a group of sleep-promoting neurons functionally, anatomically, and genetically. Overall design: Single-cell RNA-Seq of retrogradely-labeled POA neurons projecting to the tuberomammillary nucleus (TMN).

Identification of preoptic sleep-promoting neurons based on projection target

Submitted by Gene Expression Omnibus on 11-MAR-2017

Individual adult male mice (P47-66) were anesthetized in an isoflurane chamber, decapitated, and the brain was immediately removed and submerged in fresh ice-cold artificial cerebrospinal fluid (ACSF). The brain was sectioned on a vibratome (Leica VT1000S) on ice, and each slice (300-400 µm) was immediately transferred to an ACSF bath at room temperature. After the brain slicing is complete (not more than 15 minutes), individual slices of interest were transferred to a small Petri dish containing bubbled room temperature ACSF. The regions of interest were microdissected under a fluorescence dissecting microscope. The dissected tissue pieces were transferred to a microcentrifuge tube and treated with 1 mg/ml pronase in carbogen-bubbled ACSF for 70 minutes at room temperature without mixing in a closed tube. After incubation, with the tissue pieces sitting at the bottom of the tube, the pronase solution was pipetted out of the tube and exchanged with cold ACSF containing 1% fetal bovine serum. The tissue pieces were dissociated into single cells by gentle trituration through Pasteur pipettes with polished tips of 600, 300, and 150-µm diameter. Single cells were isolated by FACS into individual wells of 96-well plates or 8-well PCR strips containing 2.275 µl of Dilution Buffer (SMARTer Ultra Low RNA Kit for Illumina Sequencing, Clontech Cat#634936), 0.125 µl RNase inhibitor (SMARTer kit), and 0.1 µl of 1:1,000,000 diluted RNA spike-in RNAs (ERCC RNA Spike-In Mix 1, Life Technologies Cat#4456740). Sorting was performed on a BD FACSAriaII SORP using a 130um nozzle, a sheath pressure of 10psi, and in the single cell sorting mode. To exclude dead cells, DAPI (DAPI*2HCl, Life Technologies Cat#D1306) was added to the single cell suspension to the final concentration of 2 ng/ml. FACS populations were chosen to select cells with low DAPI and high YFP fluorescence (for SADB-rEIAV-tloop-DIO-nls-YFP injected animals) or  tdTomato (tdT) fluorescence (for Chat-IRES-Cre;Ai14 animals). Sorted cells were frozen immediately on dry ice and stored at -80°C. We used the SMARTer kit from Clontech (SMARTer Ultra Low RNA Kit for Illumina Sequencing, Clontech Cat#634936)  to reverse transcribe single cell RNA, and amplify the cDNA for 19 PCR cycles. To stabilize the RNA after quickly thawing the cells on ice, we immediately added to each sample an additional 0.125 µl of RNase inhibitor mixed with SMART CDS Primer II A. All steps downstream were carried out according to the manufacturer’s instructions. Each amplification experiment included a set of controls: 10 pg cortex RNA (isolated from Rbp4-Cre;Ai14, P57 male) as positive control for amplification, ERCC-only control to demonstrate the absence of RNases throughout the sorting process, and water-only control, to control for specificity of amplification/absence of contamination. cDNA concentration was quantified using Agilent Bioanalyzer High Sensitivity DNA chips. For most samples, 1 ng of amplified cDNA was used as input to make sequencing libraries with Nextera XT DNA kit (Illumina Cat#FC-131-1096).  We stopped the procedure after PCR clean-up and did not perform library normalization or library pooling. Individual libraries were quantified using Agilent Bioanalyzer DNA 7500 chips. In order to assess sample quality and adjust the concentrations of libraries for multiplexing on HiSeq or NextSeq. Libraries were sequenced on Illumina HiSeq single end 100-bp or NextSeq single end 75-bp.

Identification of preoptic sleep-promoting neurons based on projection target

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