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| Status |
Public on Apr 03, 2024 |
| Title |
IBA group, Subject 3 |
| Sample type |
SRA |
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| Source name |
Arcuate nucleus of hypothalamus/Median eminence
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| Organism |
Ictidomys tridecemlineatus |
| Characteristics |
tissue: Arcuate nucleus of hypothalamus/Median eminence
genotype: WT
group: Inter-bout arousal (IBA)
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| Treatment protocol |
During the active season (May – August), squirrels were kept at 20°C under a 12 hour:12 hour light:dark cycle at 40 – 60% humidity and maintained on a diet of dog food (IAMS) supplemented with sunflower seeds, superworms, and fresh vegetables (celery and carrots) with ad libitum access to water. During hibernation (September – April), hypothermic squirrels (body temperature ~20 °C) were moved to 4°C at 40 – 60% humidity under constant darkness, without access to food or water. In this study, “active” squirrels were those with a constant core body temperature (CBT) of 37°C during the active season. “IBA” squirrels were those who had undergone at least one bout of hypothermic torpor during the hibernation season, but had achieved a CBT of > 32°C for ≥ 60 minutes, or ≥ 20 minutes for the central thyroid hormone experiments.
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| Growth protocol |
All experimental procedures were performed in compliance with the Institutional Animal Care and Use Committee of Yale University (protocol 2021-11497). Thirteen-lined ground squirrels (Ictidomys tridecemlineatus; age 0.5 – 3 years) of both sexes were single housed in temperature- and humidity-controlled facilities at Yale University. All squirrels were implanted with an interscapular temperature transponder (IPTT-300, BMDS).
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Primary neurons were isolated from the arcuate nucleus of hypothalamus and median eminence following a published protocol (Vazirani et al. 2013 JOVE) with modification (n = 3). Animals were euthanized by isoflurane inhalation overdose followed by cardiac perfusion with brain perfusion solution (containing in mM: 196 sucrose, 2.5 KCl, 28 NaHCO3, 1.25 NaH2PO4, 7 Glucose, 1 Sodium Ascorbate, 0.5 CaCl2, 7 MgCl2, 3 Sodium Pyruvate, oxygenated with 95% O2/5% CO2, osmolarity adjusted to 300 mOsm with sucrose, pH adjusted to 7.4). The brain was dissected and slices were cut on a vibratome (Leica, VT1200). A brain slice containing the ARC and ME were identified by the presence of the third ventricle and separation of the optic chiasm. Three successive 600-µm slices containing the ARC were collected. The area around the third ventricle was microdissected from the brain slices using a micro-scalpel (Fine Science Tools, 10055-12). Tissue was digested in Hibernate A medium (custom formulation with 2.5 mM glucose and osmolarity adjusted to 280 mOsm, BrainBits) supplemented with 1 mM lactic acid (Sigma, L1750), 0.5 mM GlutaMAX (ThermoFisher, 35050061) and 2% B27 minus insulin (ThermoFisher, A1895601) containing 20 U/ml papain (Worthington Biochemical Corporation, LS003124) in a shaking water bath at 34°C for 30 min and dissociated by mechanical trituration through the tips of glass Pasteur pipettes with decreasing diameter (0.9 mm, 0.7 mm, 0.5 mm, 0.3 mm). Cell suspension was centrifuged over 8% bovine serum albumin (Sigma, A9418-5G) layer. Supernatant was removed leaving ~50 µl of suspension. Cell suspension was resuspended in 950 µl of Hibernate A medium (same formulation as above) and centrifuged at 300 rcf for 5 min. Supernatant was removed, leaving ~50 µl of cell suspension, which was gently mixed with a glass pipette and stored on ice. A 10 µl aliquot of cell suspension was mixed with 10 µl of Trypan Blue stain, loaded into a hemocytometer, and used to assess cell concentration and viability.
Cell suspension was processed according to the 10X Genomics library preparation protocol at the Center for Genome Analysis/Keck Biotechnology Resource Laboratory at Yale University. Single cell suspension in RT Master Mix was loaded on the Single Cell G Chip and partitioned with a pool of about 750,000 barcoded gel beads to form nanoliter-scale Gel Beads-In-Emulsions (GEMs). The volume of cell suspension for loading was calculated based on cell concentration to capture 10,000 cells. Upon dissolution of the Gel Beads in a GEM, the primers were released and mixed with cell lysate and Master Mix. Incubation of the GEMs produced barcoded, full-length cDNA from poly-adenylated mRNA. Silane magnetic beads were used to remove leftover biochemical reagents and primers from the post GEM reaction mixture. Full-length, barcoded cDNA was amplified by PCR to generate sufficient mass for library construction. Enzymatic Fragmentation and Size Selection were used to optimize the cDNA amplicon size prior to library construction. R1 (read 1 primer sequence) was added to the molecules during GEM incubation. P5, P7, a sample index, and R2 (read 2 primer sequence) were added during library construction via End Repair, A-tailing, Adaptor Ligation, and PCR. The final libraries contained the P5 and P7 primers used in Illumina bridge amplification. Sequencing libraries were sequenced on an Illumina NovaSeq instrument with 150 bp reads according to the manufacturer's instructions at the depth of ~1.1-1.4 billion reads/sample.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Raw sequencing reads were processed using 10X CellRanger v.6.1.2 (10X Genomics, Pleasanton, CA).
Custom genome reference for thirteen-lined ground squirrel (Ictidomys tridecemlineatus) was built based on the reference genome sequence and annotation obtained from the Ensembl project (www.ensembl.org (6) Release 101; all files accessed on 11/20/2020)
The gene annotation was filtered to include only protein-coding genes using cellranger mkgtf. 10X CellRanger was used to obtain transcript read counts for each cell barcode, filtered for cell barcodes called as cells based on the default parameters.
Read count matrix was further processed using R 4.2.1, RStudio 2022.02.3, and Seurat 4.1.1 (7). Non-descriptive ground squirrel gene symbols (i.e. those starting with “ENSSTOG…”) were replaced with gene symbols of mouse homolog genes, using the homolog conversion table from Ensembl. Initial set of cells/barcodes was further filtered to include only those with >= 500 features/cells, >= UMIs/cells, and <= 10% of mitochondrial genes (defined as those with gene symbol starting with “MT-“). This resulted in ~11,000-20,000 cells/sample included in the dataset for further analysis, with the sequencing depth of ~70-100k reads/cell.
Read counts were processed according to the standard Seurat analysis workflow, including normalization, identification of variable features, scaling, PCA, clustering and visualization using UMAP plots. Graphs report normalized gene expression values.
Assembly: SpeTri2.0 (GCA_000236235.1)
Supplementary files format and content: Each processed file for each sample is a tar.gz package of the "filtered_feature_bc_matrix" folder from the output of the 10X cellranger count run. Each folder contains the feature-barcode matrix in the MEX format, containing the number of UMIs associated with every feature (rows) and barcode (columns), and gzipped .tsv files with feature and barcode sequences.
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| Submission date |
Sep 05, 2023 |
| Last update date |
Apr 03, 2024 |
| Contact name |
Viktor Feketa |
| E-mail(s) |
[email protected]
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| Organization name |
Yale University School of Medicine
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| Department |
Cellular & Molecular Physiology
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| Lab |
Elena Gracheva Lab
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| Street address |
333 Cedar St SHM BE58
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| City |
New Haven |
| State/province |
Connecticut |
| ZIP/Postal code |
06520 |
| Country |
USA |
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| Platform ID |
GPL30340 |
| Series (1) |
| GSE242381 |
Hypothalamic thyroid hormone deficiency underlies reversible anorexia in a mammalian hibernator |
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| Relations |
| BioSample |
SAMN37285139 |
| SRA |
SRX21638115 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7761920_iba-squirrel3-filtered-matrix.tar.gz |
107.8 Mb |
(ftp)(http) |
TAR |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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