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Sample GSM5460430 Query DataSets for GSM5460430
Status Public on Nov 01, 2021
Title 16wk_HFD14
Sample type SRA
 
Source name 16wk HFD14
Organism Mus musculus
Characteristics cell type: cumulus cells
group: high-fat diet 16 weeks
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.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
 
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 100g/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.
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
 
Submission date Jul 17, 2021
Last update date Nov 01, 2021
Contact name Felix Krueger
E-mail(s) fkrueger@altoslabs.com
Organization name Altos Labs
Department Bioinformatics
Street address Granta Park
City Cambridge
ZIP/Postal code CB21 6GP
Country United Kingdom
 
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
Relations
BioSample SAMN20293085
SRA SRX11489305

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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