Green dutp

Mouse BAC clones encompass the locus containing Hoxb (RP23-205E11 and RP23-9G13), Ets1 (RP23-403F11 and RP23-101N3), Sept4 (RP23-333D13), or Rara (RP23-333D2 and RP23-364P11). BAC clones were verified by PCR using primers against regions of each gene. Probes were prepared by nick translation kit using green dUTP, orange dUTP, or Aqua dUTP according to the manufacturer's protocol (Abbott Molecular, Des Plaines, IL, USA). At least 100 ng of each probe was used in hybridization.

Rearrangements of ROS1 (6q22) and RET (10q11) were independently detected using a laboratory developed dual-color break-apart probe (BAP) strategy probe set (FROS1 and FLRET, Mayo Medical Labs, Rochester, MN). 5′ and 3′ of probes were labeled with either green or red fluorescence, respectively. For detecting BRAF (7q34) rearrangement, bacterial artificial chromosome (BAC) clones flanking BRAF were obtained from the Children's Hospital Oakland Research Institute (Oakland, CA). DNA isolation, nick translation, and hybridization were performed as described previously28 (link). The 5′ BRAF BACs were labeled with Green dUTP (Abbott Molecular, Des Plaines, IL) and included clones RP11-767F15, RP11-73H23, and RP11-715H9. The 3′ BAC RP11-577C22 was labeled with Orange dUTP (Abbott Molecular, Des Plaines, IL). Tumor samples were considered positive if ≥10% of 200 cells showed split signals.

FISH was employed following the protocol described previously69 (link), with some modifications. Briefly, metaphase chromosome spreads were prepared from male P. alecto primary kidney cells70 (link). DNA (1 μg) from each BAC clone isolated (Supplementary Table 3) was labelled by nick translation with Green-dUTP or Orange-dUTP (Abbott Molecular, U.S.A). 0.5–1.0 μg labelled BAC DNA, co-precipitated with 1 μg of P. alecto sheared genomic DNA, was hybridised to metaphase chromosomes and fluorescent signals were detected following the protocol described previously69 (link). A Zeiss Axio ScopeA1 epifluorescence microscope was used to visualise fluorescent signals. Images of fluorescent signals and DAPI-stained metaphase chromosomes were captured on an AxioCam MRm Rev.3 CCD (charge-coupled device) camera (Carl Zeiss Ltd, Germany) and merged using Isis FISH Imaging System version 5.4.11 (MetaSystems, Germany).

Fluorescence in situ hybridization analyses were performed on chromosome preparations generated from the cell lines using the following conventional techniques: colcemid arrest, hypotonic treatment, and methanol/glacial acetic acid fixation, as described previously [34 (link)]. The following bacterial artificial chromosome (BAC) clones were used: CH82-199H02 and CH82-179B09 for the MDM2 region, CH82-213B06 and CH82-204K11 for the CDK4 region, CH82-99P23 and CH82-60O16 for the CFA 7 region (58.4 Mb to 58.9 Mb), CH82-1E17 and CH82-40I15 for the region containing GABPBP1, and USP8 and TRPM7 amplifications (https://bacpacresources.org/, accessed on 1 July 2021). These BAC clones were labeled using green-dUTP (Abbott Molecular, Des Plaines, IL, USA) and Cy3-dCTP (Amersham Biosciences, Chalfont, UK). The slides were analyzed by an experienced cytogeneticist (FC) using a fluorescence microscope (Axioskop2, Axio Imager Z2, Zeiss, Göttingen, Germany) and Isis imaging software (Metasystems, Altlussheim, Germany). At least 100 non-overlapping tumor nuclei were examined in this study.

RNA FISH was performed as described previously (Chen et al., 2017 (link)). Cells were fixed with 4% formaldehyde in PBS for 15 min at room temperature, followed by permeabilization with 0.5% Triton X‐100, 2 mM VRC (NEB) on ice, and two times 2x SSC wash for 10 min each. Probes were first amplified with PCR using PAM expression plasmid in RNA pulldown experiment. PCR products were then precipitated by ethanol, nick‐translated and labelled with Green d‐UTP (Abbott) and nick translation kit (Abbott). For each FISH experiment, 200 μg of probe and 20 μg of yeast tRNA were lyophilized and redissolved in 10 μl formamide (Ambion), followed by denaturation at 100°C for 10 min and chilled immediately on ice. Denatured probes were mixed with hybridization buffer at 1:1 ratio. 20 μl of hybridization mix was added onto fixed cells, followed by putting coverslip on it and incubated at 37°C for 16 h in a humidified chamber. Cells were then washed twice in 2x SSC, 50% formamide; thrice in 2x SSC; and once in 1x SSC for 5 min each in 42°C. Cells were mounted by coverslip with ProLong Gold Antifade Reagent with DAPI (Invitrogen). Fluorescence images were taken in Olympus microscope FV10000 and FV10‐ASW software (version 01.07.02.02, Olympus).

Bovine whole-chromosome painting (WCP) probes were used for identification of chromosomes involved in the Robertsonian and Tandem fusions in animals analyzed in this study. Bovine whole chromosomes were isolated by flow sorting using MoFlo XDP Cell Sorter (Beckman Coulter, Brea, CA, USA) [29 (link)] or microdissected by PALM Microlaser system (Carl Zeiss MicroImaging GmbH, Munich, Germany) [30 (link)]. Once isolated, bovine chromosomes were used to produce WCP probes by DOP-PCR [31 (link)]. Probe labeling was performed during the secondary PCR with Green-dUTP or Orange-dUTP (Abbott Park, Chicago, IL, USA) [30 (link)].
For sperm-FISH, bovine BAC clones localized to the chromosomes involved in translocations were selected from the CHORI-240 cattle library (BACPAC Genomics, Emeryville, CA, USA). BAC DNA labeling was with digoxigenin-11-dUTP or biotin16-dUTP (Roche, Mannheim, Germany) was performed using BioPrime Array CGH Genomic Labeling Module (Invitrogen, Carlsbad, CA, USA). Detailed list of BACs used in the present study appears in Table S1.