PGCs from E13.5 male embryos were sorted by flow cytometry and used for Affymetrix microarray analysis. Three individual samples of 10 000 PGCs (pooled from at least five embryos for each sample) were processed for WT and KO genotypes. PGCs were directly sorted by flow cytometry in lysis buffer from the
RNeasy micro kit (Qiagen, Cortaboeuf, France). The quantity and quality of RNA were analyzed using a
21 000 Bioanalyzer (Agilent Technologies, Les Ulis, France). Only samples with an RNA integrity number (RIN) equal to or above 7 were used for the transcriptomic analysis. RNA samples were then analyzed using an
Affymetrix GeneChip™ Mouse gene 2.0 ST Array (Thermofisher Scientific, Villebon sur Yvette, France). Our data were then studied with GSEA and Ingenuity Pathway Analysis software. For
quantitative RT-PCR, mRNA was prepared using
RNeasy® Micro and Mini kits (Qiagen, Cortaboeuf, France). The mRNA was then reverse-transcribed with a
Quantitect kit (Qiagen, Cortaboeuf, France). Quantitative RT-PCR was performed using an
AB7900 device (Applied Biosystems, Thermofisher Scientific, Villebon sur Yvette, France) with
Fast SYBR® Green Master Mix (Applied Biosystems, Thermofisher Scientific, Villebon sur Yvette, France). The primers are listed in
Supplementary Material, Table S3.
Jarysta A., Riou L., Firlej V., Lapoujade C., Kortulewski T., Barroca V., Gille A.S., Dumont F., Jacques S., Letourneur F., Rosselli F., Allemand I, & Fouchet P. (2021). Abnormal migration behavior linked to Rac1 signaling contributes to primordial germ cell exhaustion in Fanconi anemia pathway-deficient Fancg−/− embryos. Human Molecular Genetics, 31(1), 97-110.
