2024-09-22
ENA first public
ERC000011
ENA-CHECKLIST
SAMEA114526679
External Id
Junior Research Group Somatic Evolution and Early Detection German Cancer Research Center (DKFZ), Heidelberg, Germany
INSDC center name
2023-10-23T17:45:45Z
INSDC last update
public
INSDC status
ERS16508015
SRA accession
E-MTAB-13474:no3moc73
Submitter Id
3
month
age
ArrayExpress
broker name
oocyte
cell_type
not collected
collection date
house mouse
common name
Protocols: 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. Ovulation by hormonal stimulation was induced by intraperitoneal injection of 5 IU pregnant mare serum gonadotropin (PMSG - Pregmagon®) and second injection of 5 IU human chorionic gonadotropin (hCG - Ovogest®) 48 hours later (Luo et al., 2011). The COCs were collected from ampullas 14-16h after hCG injection in young mice and 18-19h in old mice. Natural ovulation was induced by mating C57BL/6 females with vasectomized CD-1 males. The mice were caged 1:1 female-male ratio shortly before the beginning of the dark cycle. The next morning females presenting a copulatory plug were sacrificed for cell collection. Young mice were sacrificed 2-3 h and old mice around 6 h after the beginning of the light cycle. Females without plugs were examined each morning for three consecutive days. Males were allowed to rest for at least one day between matings. 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. 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.
description
adult
developmental stage
wild type genotype
genotype
not collected
geographic location (country and/or sea)
NO30
individual
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
female reproductive system
organism part
Mus musculus
scientific_name
female
sex
natural ovulation
stimulus
C57BL/6J
strain
no3moc73
title