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Ribo-depleted total RNA-seq of oocytes in 3M mice after natural and superovulation

ABSTRACT: Ribo-depleted total RNA-seq of oocytes in 3M mice after natural and superovulation

PROVIDER: PRJEB67724 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR12142500_1.fastq.gz	Fastqsanger.gz
	ERR12142500_2.fastq.gz	Fastqsanger.gz
	ERR12142501_1.fastq.gz	Fastqsanger.gz
	ERR12142501_2.fastq.gz	Fastqsanger.gz
	ERR12142502_1.fastq.gz	Fastqsanger.gz

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Ribo-depleted total RNA-seq of oocytes in 3M mice after natural and superovulation

Project description:Two major factors contributing to reduced fertility is use of exogenous hormones and old age. We use mouse model to study transcriptional and cell-cell communication changes upon superovulation and ageing in female reproductive cells - oocytes (OC) - and somatic cells - granulosa (GC) - surrounding them. Here, we are validating the results obtained by a polyA-biased method. Oocytes from naturally and superovulated mice were collected as follows: mice were sacrificed by cervical dislocation and oviducts dissected. Ampullas were torn to release the COCs into a 96 µl M2 media drop under mineral oil at room temperature. Then, 4ul of pre-heated 500 µg/ml hyaluronidase diluted in M2 media (final concentration in the drop 20 µg/ml) was added to separate the COCs into single units. The COCs were incubated for 10-20 min at 37oC and then mechanically separated into individual M2 drops using 115-124 μm glass retransfer pipette. Individual COCs were then washed in M2 once and incubated for less than 5 min with enzymatic mix Accutase at 37oC to further separate granulosa cells from the oocytes. Oocytes were washed twice with M2 media and once with DPBS before collection. Cells were immediately flash frozen in liquid nitrogen in individual 0.2ml thin wall PCR tubes and stored in -80oC until library preparation. SMARTer Stranded Total RNA - Low Input Mammalian kit was used for lysis, cDNA amplification and library preparation with 10 cycles for PCR 1 and 16 cycles for PCR 2. Samples were sequenced as recommended by the manufacturer on Novaseq600 with paired-end sequencing.

2024-09-22 | E-MTAB-13474 | biostudies-arrayexpress

RNA-seq of paired oocytes and granulosa cells from 3M and 12M old mice after natural and superovulation

Project description:Oocytes and granulosa cells were collected after natural and superovulation in young (3 month-old) and old (12 month-old) mice while conserving pairing information between oocyte and granulosa cells from each cumulus-oocyte-complex (COC). This experimental design allows for the analysis of the effects of ageing and / or superovulation on the transcriptome of COCs. The naming convention for samples is as follows: [ovulation, NO/SO][age, 3M/12M][cell, OC/GC][COC number], meaning that NO12MOC01 and NO12MGC01 are from the same COC. Oocyte and granulosa cell samples were processed for full-length cytoplasmic RNA amplification using slightly modified SMART-seq2 protocol. After amplification of cDNA and library preparation, samples were sequenced on NextSeq 550 using Single-Ended 75bp High-Output kit.

2024-09-22 | E-MTAB-13479 | biostudies-arrayexpress

RNA-seq of paired oocytes and granulosa cells from 3M and 12M old mice after natural and superovulation

Project description:RNA-seq of paired oocytes and granulosa cells from 3M and 12M old mice after natural and superovulation

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Genome-wide DNA methylation assessment in mouse oocytes: effects of in vitro growth, superovulation and sexual immaturity

Project description:There is legitimate concern over epigenetic abnormalities that could be induced by procedures applied during assisted reproduction, necessitating epigenetic assessment of new methodologies. The effect of in vitro follicle culture (IFC) and superovulation on DNA methylation in the oocyte is not fully known. Here, we present the first genome-wide analysis of oocyte DNA methylation in IFC, superovulated mouse oocytes and, uniquely, we compare these data with naturally ovulated counterparts. Globally, the distinctive methylation landscape of oocytes, comprising alternating hyper- and hypomethylated domains, is preserved irrespective of the procedure. The conservation of methylation extends to the germline differential methylated regions (DMRs) of imprinted genes, necessary for their monoallelic expression in the embryo after fertilisation. However, we do detect specific, consistent and coherent differences in DNA methylation between IFC and oocytes from other groups, and between oocytes obtained after superovulation from pre-pubertal compared with sexually mature females. Several methylation differences span entire transcription units, consistent with effects on methylation downstream of transcriptional differences. Amongst these, we found alterations in Sox5, Zfp521 and Tcf4, genes for transcripton factors related to nervous system development. These observations also suggest transcriptional and epigenetic differences between oocytes from the initial and later phases of follicle recruitment. Our results provide an important reference for the use of in vitro growth and maturation, particularly from prepubertal females, in assisted reproductive technologies or fertility preservation.

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