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Transcriptome profiling of the mouse suprachiasmatic nucleus at single cell resolution

ABSTRACT: Circadian rhythms are governed by the hypothalamic suprachiasmatic nucleus (SCN) which consists of ca. 20,000 cellular oscillators bound together in a powerful time-keeping network. The goal of this study was to characterise its component cells and network structure by conducting single-cell sequencing of SCN organotypic slices. scRNASeq libraries were prepared using 10x Genomics Chromium Single Cell 3’ Library and Gel Bead v2 kit. SCN cells, diluted in 0.04% BSA-nuclease-free water, reverse transcription master mix and partitioning oil, were loaded on a ChromiumTM Single Cell A Chip and run on the Chromium Controller to obtain at target cell recovery rate of 8,000 cells. Final libraries were sequenced on one flow cell of an Illumina HiSeq 4000 with a read length of 26bp for read 1 (cell barcode and unique molecule identifier and sample barcode) and 98bp for read 2 (RNA read) to yield approximately 340 million reads per sample. We sequenced 13,324 cells from 52 SCN slices at circadian time 7.5 (mid-day) over three independent sequencing runs, and 16,996 cells from 36 SCN slices at circadian time 15.5 (mid-night) over two independent sequencing runs.

ORGANISM(S): Mus musculus

PROVIDER: GSE167927 | GEO | 2021/09/06

REPOSITORIES: GEO

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Project description:The timing of daily M-bM-^@M-^\circadianM-bM-^@M-^] behavior can be highly variable among different individuals, and twin studies suggest that about half of this variability is environmentally controlled. Similar plasticity can be seen in mice exposed to an altered lighting environment M-bM-^@M-^S for example, 22-hour days instead of 24-hour ones M-bM-^@M-^S which stably alters the genetically determined period of circadian behavior for months. The mechanisms mediating these environmental influences are unknown. Here, we show that transient exposure of mice to such lighting stably alters global transcription in the suprachiasmatic nucleus of the hypothalamus (the SCN, the M-bM-^@M-^\master clockM-bM-^@M-^] tissue determining circadian behavior in mammals). We have also showed that, these changes in transcription are due to change in DNA methylation in the SCN. Indeed, genome-wide methylation profiling revealed global alterations in promoter DNA methylation in the SCN. Importantly, infusion of a methyltransferase inhibitor to the SCN during 22-hour days suppressed period changes. We also found that these behavioral and DNA methylation changes are reversible upon entrainment to 24-hours days. We conclude that the SCN utilizes DNA methylation as a mechanism to drive circadian clock plasticity. Methods: First, 16 mice 5 to 6 weeks old were exposed to DD for 7 days in order to measure their FRP. Then, mice were separated into 2 groups of 8 and entrained to ST and NT cycle. After 4 weeks we recorded wheel-running behavior in constant darkness. After 5 days in DD, we used Clocklab software to predict time of activity onset for each mouse. Brains were isolated at CT4 and sliced with a microtome using a fresh HBSS medium (thickness 250 uM) and the SCN was cut out with the aid of a binocular microscope and stored immediately at M-bM-^@M-^S80M-BM-0C. Total RNA was extracted using the SurePrepM-bM-^DM-" Nuclear or Cytoplasmic RNA Purification Kit (Fisher Scientific) according to the manufacturer's protocol. A total of 500 ng of RNA was used for subsequent steps. These steps included DNAase treatment, polyA selection, depletion of ribosomal RNA, overall quality check, and library preparation, and were performed by Fasteris (CH). Libraries were prepared according to standard Illumina protocols. sequence quality, read mapping and data analysis: all steps were done using the GeneProf platform. Briefly, the sequencing reads of each library were first quality checked for read length and nucleotide composition, and then submitted for mapping to the mouse genome refrence sequence (Ensemble 58 mouse Genes, NCBIM37 assembly) using the alignment tool TopHat. Results: Entrainment to non-24 hours light/dark cycle induces a global changes in the SCN transcriptome. SCN mRNA profile of mice entrained to normal and short T-cycle using RNAseq technology.

Project description:To screen for specific circadian outputs that may distinguish the pacemaker in the mammalian suprachiasmatic nucleus (SCN) from peripheral-type oscillators in which the canonical clockworks are similarly regulated in a circadian manner, the rhythmic behavior of the transcriptome in forskolin-stimulated NIH/3T3 fibroblasts was analyzed and compared to that found in the rat SCN in vivo and SCN2.2 cells in vitro. Similar to the scope of circadian gene expression in SCN2.2 cells and the rat SCN, NIH/3T3 fibroblasts exhibited circadian fluctuations in the expression of the core clock genes, Per2, Bmal1 (Mop3), and Cry1 and 323 functionally diverse transcripts (2.6%), many of which were involved in cell communication. Overlap in rhythmically-expressed transcripts among NIH/3T3 fibroblasts, SCN2.2 cells and the rat SCN was limited to these clock genes and four other genes that mediate fatty acid and lipid metabolism or function as nuclear factors. Compared to NIH/3T3 cells, circadian gene expression in SCN oscillators was more prevalent among cellular pathways mediating glucose metabolism and neurotransmission. Coupled with evidence for the rhythmic regulation of the inducible isoform of nitric oxide synthase, the enzyme responsible for the production of nitric oxide, in SCN2.2 cells and the rat SCN but not in fibroblasts, studies examining the effects of a NOS inhibitor on metabolic rhythms in co-cultures containing SCN2.2 cells and untreated NIH/3T3 cells suggest that this gaseous neurotransmitter may play a key role in SCN pacemaker function. Thus, this comparative analysis of circadian gene expression in SCN and non-SCN cells may have important implications in the selective identification of circadian signals involved in the coupling of SCN oscillators and the regulation of rhythmicity in downstream cells or tissues. Experiment Overall Design: Circadian profiling of the NIH/3T3 fibroblast transcriptome entailed the treatment of NIH/3T3 cells with a 15uM forskolin pulse, subsequent washout of the drug, and collection of total RNA immediately after washout and every 6 hours across two circadian cycles for each of three experiments. Timepoint values reflect the average of three samples from these biological replicates.

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Transcriptome profiling of the mouse suprachiasmatic nucleus at single cell resolution

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