Nick translation kit

Clone 395, derived from the library representing the most frequently present sequences in the F. pratensis genome, was labeled by nick translation with fluorochrome Atto647 (Jena BioScience) (Majka et al. 2017b (link)). The ribosomal sequence 35S rDNA was labeled with digoxigenin-11-dUTP by nick translation. While 5S rDNA and the Arabidopsis thaliana L. telomere repeats (TTTAGGG)_n were labeled by polymerase chain reaction (PCR) with tetramethyl-rhodamine-5-dUTP (Sigma). The total genomic DNA of L. perenne was used as a probe for genomic in situ hybridization and was labeled with digoxigenin-11-dUTP using nick translation kit according to manufacturer instruction (Sigma).

RNA-DNA FISH was performed using a modification of the previously published method (Nath and Johnson 2009 (link); Barakat and Gribnau 2014 (link); Lai et al. 2016 ). The RNA FISH probe was prepared as a short antisense single-strand DNA that crosses the introns to probe for the mature RNAs (Zhao et al. 2019 (link)). Briefly, the short single-strand DNA probe was synthesized using a ratio of 1:50 primers that cover the intron splicing site with Dig labeling dNTP MIX (Roche #11277065910). The Sox2 DNA probe was prepared from Sox2 BAC clone RP23-213M12 (Bacpacresources.org) with biotin-14-dCTP using a nick translation kit (Sigma-Aldrich) according to the manufacturer's protocol. After sequential RNA and DNA FISH, slides were counterstained with DAPI. The FISH images were collected with Chroma filter sets using an Olympus BX41 upright microscope (100×, oil, 1.4 NA) equipped with a motorized z-axis controller (Prior) and Slidebook 5.0 software (Intelligent Imaging Innovations). The optical sections were collected using a NoNeighbor algorithm operating within Slidebook 5.0. The geometric centers of foci were quantitated in Slidebook 5.0. Images were merged to confirm the colocalization of the DNA-RNAs.

FISH was performed as previously described (Silva et al., 2014 (link)). Gonads of young adult worms were dissected and fixed in 7.4% paraformaldehyde. After washing three times in 2× SCCT (1x saline-sodium citrate buffer [SSC] with 0.1% Tween-20) buffer, gonads were dehydrated by incubation in increasing ethanol concentrations and air-dried at room temperature. Chromosome III containing the pairing center (cosmid T17A3) and the chromosome V 5S ribosomal DNA (rDNA) locus labeled with biotin and digoxigenin, respectively, were used as FISH probes according to the manufacturer’s instructions for the Nick Translation Kit (Sigma-Aldrich). Denaturation and annealing were performed on a heat block for 3 min at 93°C, 2 min at 72°C, and overnight at 37°C. Afterward, slides were washed once at 37°C with a 1:1 ratio of 2× SCCT: 50% formamide, once in 2× SCCT containing 10% Tween-20 and then three times in 2× SCCT. Slides were then blocked in 1% BSA, and fluorescently labeled anti-biotin and anti-digoxigenin antibodies were applied for 1 h at room temperature. After DAPI staining, the slides were mounted in Vectashield.