Gotaq flexi reaction buffer

Probes for fluorescence in situ hybridization corresponding to CgiSat02, 03, 04, 05, 09, 17, 28, 37, 46 and 5S rDNA were labeled by PCR. The reactions contained 50 ng of DNA, 100 µM dATP, dGTP and dCTP, 65 µM dTTP, 2.5 mM MgCl2, 2.5 U GoTaq G2 Flexi Taq DNA polymerase, 1× GoTaq Flexi Reaction Buffer (all Promega, Madison, WI, USA), primers (1 µM each) and 35 µM biotin-16-dUTP (Jena Bioscience, Jena, Germany) for satDNAs or 35 µM digoxigenin-16-dUTP (Roche, Basel, Switzerland) for 5S rDNA, in 50 µL volumes. Nucleotide sequences of each primer pair used and PCR amplification conditions employed are presented in Table S5. Probe purification was performed using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany), following the protocol within. Probes were checked on 1% agarose gel and the concentration of the purified probes was measured using a Qubit Fluorometer. 30 ng of probe was used per FISH experiment.

Probes for fluorescent in situ hybridization (FISH) of sequences belonging to Cg170, Cl112, Cl150, Cl344, Cl460 and Cl485 were PCR-labelled in 50 µL volumes, containing 250 ng of DNA, 50 µM dATP, dGTP and dCTP, 32 µM dTTP and 18 µM biotin-16-dUTP, primers (1 µM each), 2.5 mM MgCl2, 2.5 U GoTaq G2 Flexi Taq DNA polymerase and 1× GoTaq Flexi Reaction Buffer (all Promega). The initial denaturation was performed at 94 °C for 5 min and was followed by 30 cycles of amplification under the conditions shown in Table S1 for each set of primers. The final extension was done at 72 °C for 7 min. Probes were purified using QIAquick PCR Purification Kit (Qiagen) according to the manufacturer’s protocol. Probes were checked on 1% agarose gel for quality and the concentration of the purified probes was measured using a Qubit Fluorometer. Probes for the detection of CenH3- and H3K9me3-associated sequences were biotin-labelled by the random priming method, using 66 ng and 48 ng of ChIP-ed DNA, respectively.