2024-09-22
ENA first public
ERC000011
ENA-CHECKLIST
SAMEA114526503
External Id
Junior Research Group Somatic Evolution and Early Detection German Cancer Research Center (DKFZ), Heidelberg, Germany
INSDC center name
2023-10-23T17:36:42Z
INSDC last update
public
INSDC status
ERS16507839
SRA accession
E-MTAB-13479:SO12MOC22
Submitter Id
12
month
age
ArrayExpress
broker name
oocyte
cell_type
not collected
collection date
house mouse
common name
Protocols: Mice were sacrificed by cervical dislocation and oviducts dissected. Under the stereomicroscope 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. When separated, a small bulk of GCs (~50-200) were collected from these individual drops using a mouth pipette pre-filled with Dulbecco's phosphate-buffered saline. 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 further use. 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. 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. Oocyte and granulosa cell samples were processed for full-length cytoplasmic RNA amplification using slightly modified SMART-seq2 protocol. Briefly, cells were lysed with 0.2% Triton X-100 and reverse transcription was facilitated by Maxima-H Minus Reverse Transcriptase. Amplification of complementary deoxyribonucleic acid (cDNA) was achieved with 17 PCR cycles for oocytes and 20 cycles for granulosa cells. Libraries for next generation sequencing were prepared using Nextera XT DNA library Prep Kit. 1 ng of cDNA was used as template for library preparation, then tagmented, double indexed and amplified with 11 PCR cycles. All libraries were multiplexed into a single pool prior to sequencing. Quality control for sample preparation was performed using 4200 TapeStation High Sensitivity DNA and D1000 tapes, and Qubit 4 fluorometer High Sensitivity dsDNA assays.
description
adult
developmental stage
wild type genotype
genotype
not collected
geographic location (country and/or sea)
SO44
individual
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
female reproductive system
organism part
Mus musculus
scientific_name
female
sex
superovulation
stimulus
C57BL/6J
strain
SO12MOC22
title