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Sample GSM5460426

Query DataSets for GSM5460426

Status

Public on Nov 01, 2021

Title

16wk_CD9

Sample type

SRA

Source name

16wk CD9

Organism

Mus musculus

Characteristics

cell type: cumulus cells
group: chow 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