Rolling circle amplification (RCA) FISH

RCA FISH was performed according to [41] protocol with the following changes: oocytes were fixed 10 min in 4% PFA (Sigma Aldrich) and permeabilized in 0.1% Triton X-100 in PBS for 10 min and subsequently in 70% ethanol, pre-frozen to -20°C, for 10 seconds. The whole transcriptome was converted into cDNA by M-MuLV reverse transcriptase (Enzymatics) and the reaction mix was prepared according to the mentioned protocol. The cDNA fragments were fixed to the cellular protein matrix using a nonreversible amine cross-linker BS(PEG)9 (Sigma Aldrich) and circularized after degrading the RNA residues. The circular templates were amplified using RCA primers 100 μM (TCTTCAGCGTTCCCGA*G*A; where * is phosphorothioate, Generi Biotech) complementary to the adapter sequence in the presence of aminoallyl-dUTP and stably cross-linked. For visualization of endogenous cDNA we used fluorescently labeled random octamers, which were labeled by two fluorophore (green-488 nm) or (red-561 nm). Chromatin was visualized by incubation (1min) with 10 nM DAPI (Sigma Aldrich) in 2xSSC; then washed 1x with 2xSSC. We scanned the samples in 2xSSC. Quantification of fluorescent intensity between cytoplasm and nucleus was used ImageJ/FIJI (http://rsbweb.nih.gov/ij/). Per one experiment was used 12 oocyte and 10 embryos, we quantified 3 experiments.