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RNA-seq of paired granulosa cells and morulas or blastocysts from naturally and superovulated 3M old mice

ABSTRACT: RNA-seq of paired granulosa cells and morulas or blastocysts from naturally and superovulated 3M old mice

PROVIDER: PRJEB77920 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR13385800.fastq.gz	Fastqsanger.gz
	ERR13385900.fastq.gz	Fastqsanger.gz
	ERR13385701.fastq.gz	Fastqsanger.gz
	ERR13385801.fastq.gz	Fastqsanger.gz
	ERR13385901.fastq.gz	Fastqsanger.gz

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RNA-seq of paired granulosa cells and morulas or blastocysts from naturally and superovulated 3M old mice

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. In this experiment, we are testing how different GC transcriptional profiles correlate with embryo quality. 3M old C57BL6/J mice were naturally or superovulated and COCs singularized as described above. Granulosa cells were immediately flash frozen and matched oocytes were divided into individual drops of CARD media under mineral oil for fertilization. The oocytes were incubated in 37oC, 5% CO2 incubator for 30-40 min prior to fertilization. Frozen Mus musculus (C57BL6/J) male sperm straws were thawed and capacitated in Fertiup media for 30 min. Fertilization was achieved by combining 2.5 µl of activated sperm with 20 µl CARD media drop containing a single oocyte and incubated at 37oC, 5% CO2 for 3 hours. Then, oocytes were individually washed in Human Tubal Fluid (HTF) media drops and transferred to 20 µl HFT drops for overnight culture. After 24 hours, fertilization rate was evaluated by 2-cell stage and embryos transferred to G1+ media for further development. Early morula stage embryos were continuously collected based on observation 62-64 h post-fertilization. For late blastocysts, fertilization media was additionally changed to G2+ after 48h post- fertilization and blastocysts collected at 108h post-fertilization. For collection, the embryos were washed in DPBS and flash frozen. Granulosa cells and embryo samples were further processed by Smart-seq2, the cDNA amplification PCR cycle number for embryos was 16-17.

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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.

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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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Project description:Fresh human HCC tissue and paired pertumor liver tissue were acquired, isolation of the infiltrating lymphocytes was conducted by discontinous density gradient centrifugation in Percoll. After removal of dead cells and incubation with components of TCRM-NM-3/M-NM-4 isolation kit (Mitenyi Biotec), M-NM-3M-NM-4 T cells were positively selected from the single-cell suspension using autoMACS Separator (Miltenyi Biotec) with recommended programs. Then we employed whole genome microarray expression profiling as a discovery platform to indentify genes which are differentially expressed between HCC-derived and paired peritumor-derived M-NM-3M-NM-4T cells. Ten paired fresh HCC tissue and peritumor liver tissue were acquired and subjected to M-NM-3M-NM-4T cells isolation.

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