GSM2610532: PGC12M; Mus musculus; miRNA-Seq
Mus musculus CD1 strain was used as animal model. PGCs and somatic gonadal cells were isolated from embryos obtained from pregnant females at E11.5, E12.5 and E13.5. At E11.5 and E12.5, male and female gonads are morphologically indistinguishable at early stages of development. To sex embryos at E11.5 and E12.5, we isolated and stored individually each set of gonads in PBS at 4ºC. Sex was identified in each single embryo by PCR of DNA, obtained from the heads, using primers to identify the presence of Sry gene and Jarid1d (a single band for XX and a double band for XY). After sexing the embryos, the mesonephros was removed before the purification of germ and somatic cells. PGCs and somatic cells were purified using a paramagnetic technology according to Brieño-Enriquez et al. 2015. Briefly, groups of about 80 gonads were incubated in 0.25% trypsin-EDTA during 15 minutes at 37ºC, washed followed by incubation with PGC-specific antibodies (anti-CD15) bound to paramagnetic-microbeads. Finally, PGCs were isolated from somatic stromal cells by a magnetic column (miniMAcs) and stained both separated fractions with the naphtol AS-MX/ FAST-RED (Sigma-Aldrich) (PGC specific) to determine the level of enrichment of cell populations. In all cases, the enrichment of the PGCs was 93-96%. Similarly, the purity of somatic cells was over 90% in all samples. RNA libraries were prepared for sequencing using standard Illumina protocols.