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

ABSTRACT: 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.

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Mus musculus

SUBMITTER: Angela Gonçalves 

PROVIDER: E-MTAB-13474 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress