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Status |
Public on Nov 01, 2021 |
Title |
16wk_HFD_lowg22 |
Sample type |
SRA |
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Source name |
16wk HFD lowg22
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Organism |
Mus musculus |
Characteristics |
cell type: cumulus cells group: high-fat diet 16 weeks low weight gainer
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Extracted molecule |
total RNA |
Extraction protocol |
Cumulus cells were collected into RLT buffer (1053393, Qiagen, Hilden, Germany) and kept in -80oC until library generation. mRNA was captured using Smart-seq2 oligo-dT pre-annealed to magnetic beads (MyOne C1, Invitrogen, Carlsbad, California, USA). The beads were resuspended in 10 μl of reverse transcriptase mix (100 U, SuperScript II, Invitrogen; 10 U, RNAsin, Promega, Madison, Wisconsin, USA), 1 × Superscript II First-Strand Buffer, 2.5 mM ditiotreitol (DTT, Invitrogen), 1 M betaine (Sigma Aldrich), 9 mM magnesium chloride (MgCl2, Invitrogen), 1 μM Template-Switching Oligo (Exiqon, Vedbaek, Denmark), 1 mM dNTP mix (Roche) and incubated for 60 min at 42 °C followed by 30 min at 50 °C and 10 min at 60 °C (Picelli et al., 2013, 2014). Amplification of the cDNA was then undertaken after adding 11 μl of 2 × KAPA HiFi HotStart ReadyMix and 1 μl of 2 μM ISPCR primer (Picelli et al., 2013, 2014), followed by the cycle: 98 °C for 3 min, then 9 cycles of 98 °C for 15 s, 67 °C for 20 s, 72 °C for 6 min and finally 72 °C for 5 min. Finally, the cDNA was purified using similar volume of AMPure beads (Beckman Coulter, Brea, California, USA) and eluted into 20 μl of water. All libraries were prepared from 100 to 200 pg of cDNA using the Nextera XT Kit (Illumina, San Diego, California, USA), regarding the manufacturer’s instructions. The final cDNA library was purified using a 0.7:1 volumetric ratio of AMPure beads before pooling and sequencing.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NextSeq 500 |
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Description |
Breeding pairs of C57BL/6J (B6) mice were obtained from the Jacksons Laboratory (The Jacksons Laboratory, Bar Harbor, Maine, USA). At 8 weeks (wk) of age B6 animals were divided into 2 groups (n=10/group). In DIO, the control group was fed ad libitum chow diet (CD, 11% energy in kcal from fat, 5053, rodent diet 20, LabDiet IPS, London, UK), while the experimental group received high fat diet (HFD, 58% energy in kcal from fat, AIN-76A 9G03, LabDiet IPS). Mice were maintained under the diet for 4 or 16 wk. Regarding the pharmacological hyperleptinemia protocol (LEPT), 8 wk old B6 female mice were divided into 4 groups (n=15/group): i) saline 9 days (d); ii) leptin 9 d; iii) saline 16 d; and iv) leptin 16 d (Recombinant Mouse Leptin, GFM26, Cell Guidance Systems, Cambridge, UK). The animals were injected intraperitoneally twice a day, at 09:00h and 21:00h and total dosage of 100g/day of leptin was administrated. For all protocols, mice were housed with a 12h light/12h dark cycle at room temperature. A total of approximately 50 cumulus cells were collected from one individual cumulus oophorous complex, obtained after superovulation of female mice from either 4 wk and 16 wk DIO or 16 d leptin protocols.
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Data processing |
Trim galore v0.4.2 was used with default parameters on raw fastq sequence files. Mapping of the RNA-seq data was done with HISAT2 v2.0.5 against the mouse GRCm38 genome, as guided by known splice sites taken from Ensembl v68. Genome_build: GRCm38 Matrix (.txt) of raw counts
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Submission date |
Jul 17, 2021 |
Last update date |
Nov 01, 2021 |
Contact name |
Felix Krueger |
E-mail(s) |
fkrueger@altoslabs.com
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Organization name |
Altos Labs
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Department |
Bioinformatics
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Street address |
Granta Park
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City |
Cambridge |
ZIP/Postal code |
CB21 6GP |
Country |
United Kingdom |
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Platform ID |
GPL19057 |
Series (1) |
GSE180300 |
Leptin Signalling in the Ovary of Diet-Induced Obese Mice Regulates Activation of Nod-Like Receptor Protein 3 Inflammasome |
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Relations |
BioSample |
SAMN20293114 |
SRA |
SRX11489310 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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