Key resources table.
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| Rabbit anti-RAD51 | Abcam | Cat#ab176458; RRID: AB_2665405 |
| Rabbit anti-RAD51 (7946) | homemade | N/A |
| Rabbit anti-Lamin-A | Sigma-Aldrich | Cat#L1293; RRID: AB_532254 |
| Rabbit anti-CENP-B | Bethyl Laboratories | Cat#IHC-00064; RRID: AB_669682 |
| Mouse anti-CENP-A | Abcam | Cat #ab13939; RRID: AB_300766 |
| Mouse anti-CENP-A | GeneTex | Cat#GTC13939; RRID: AB_369391 |
| Rabbit anti-TOP1 | Abcam | Cat#ab109374; RRID: AB_10861978 |
| Rabbit anti-TOP2A | Cell Signaling Technology | Cat#12286; RRID: AB_2797871 |
| Rabbit anti-TOP2B | GeneTex | Cat#GTX102640-GTX-25ul; RRID: AB_11169314 |
| Rabbit anti-TOP3A | Proteintech | Cat#14525-1-AP; RRID: AB_2205881 |
| Rabbit anti-TOP3B | Biorbyt | Cat#ORB127293 |
| Mouse anti-Tubulin | Cell Signaling Technology | Cat#3873; RRID: AB_1904178 |
| Rabbit anti-Histone H3 | Bethyl Laboratories | Cat#A300-823A-T; RRID: AB_2118462 |
| Goat anti-rabbit Alexa Fluor 488 | Thermo Fisher Scientific | Cat#A11070; RRID: AB_142134 |
| Goat anti-rabbit Alexa Fluor 555 | Thermo Fisher Scientific | Cat#A21430; RRID: AB_1500773 |
| Goat anti-rabbit Alexa 647 | Thermo Fisher Scientific | Cat#A27040; RRID: AB_2536101 |
| Goat anti-mouse Alexa Fluor 488 | Thermo Fisher Scientific | Cat#A11017; RRID: AB_143160 |
| Goat anti-mouse Alexa Fluor 555 | Thermo Fisher Scientific | Cat#A21425; RRID: AB_1500751 |
| Goat anti-mouse Alexa 647 | Thermo Fisher Scientific | Cat#A21237; RRID: AB_1500743 |
| Goat anti-mouse HRP-conjugated | Agilent | Cat#P0447; RRID: AB_2617137 |
| Goat anti-rabbit HRP-conjugated | Agilent | Cat#P0448; RRID: AB_2617138 |
| Chemicals, peptides, and recombinant proteins | ||
| Exonuclease III | Promega | Cat#M1811 |
| Aphidicolin | Santa Cruz Biotechnology | Cat#sc-201535 |
| Lipofectamine RNAiMAX | Thermo Fisher Scientific | Cat#13778075 |
| FISH blocking solution | Roche | Cat#11096176001 |
| BsmAI | NEB | Cat# R0529S |
| Nt.BsmAI | NEB | Cat#R0121S |
| Critical commercial assays | ||
| TURBO DNA-free kit | Invitrogen | Cat#AM1907 |
| cDNA reverse transcription kit | Applied Biosystems | Cat#4368814 |
| Deposited data | ||
| Raw and analysed data | This paper | Mendeley Data DOI: 10.17632/65jt7xwr2p.1 |
| Raw sequencing data | This paper | PRJNA885500 |
| T2T-CHM13 reference genome | GitHub | https://github.com/marbl/CHM13 |
| Experimental models: Cell lines | ||
| hTERT-RPE1 | ATCC | RRID:CVCL4388 |
| hTERT-RPE1 Flp-In T-REx | Jonathon Pine | CancerTools.org:Cat#153242 |
| HCT116 | ATCC | RRID:CVCL0291 |
| HeLa | ATCC | RRID:CVCL0030 |
| Oligonucleotides | ||
| Universal negative control siMisNeg | Merck | Cat#SIC001 |
| siRAD51 #1 (5’ GACUGCCAGGAUAAAGCUU 3’) |
Custom order: Integrated DNA Technologies (IDT) | N/A |
| siRAD51 #2 (5’ GUGCUGCAGCCUAAUGAGA 3’) |
Custom order: Integrated DNA Technologies (IDT) | N/A |
| siRAD51 3’UTR #1 (5’ GACUGCCAGGAUAAAGCUU 3’) |
Custom order: Integrated DNA Technologies (IDT) | N/A |
| siRAD51 3’UTR #2 (5’ GUGCUGCAGCCUAAUGAGA 3’) |
Custom order: Integrated DNA Technologies (IDT) | N/A |
| siCENP-A (5’ GGACUCUCCAGAGCCAUGAUU 3’) |
Custom order: Integrated DNA Technologies (IDT) | N/A |
| Silencer Select RNAi targeting TOP1 | Thermo Fisher Scientific | Cat#4390824; siRNA ID s14305 |
| Silencer Select RNAi targeting TOP2A | Thermo Fisher Scientific | Cat#4390824; siRNA ID s14308 |
| Silencer Select RNAi targeting TOP2B | Thermo Fisher Scientific | Cat#4390824; siRNA ID s106 |
| Silencer Select RNAi targeting TOP3A | Thermo Fisher Scientific | Cat#4390824; siRNA ID s14311 |
| Silencer Select RNAi targeting TOP3B | Thermo Fisher Scientific | Cat#4390824; siRNA ID s17099 |
| II3A mutagenesis primers (R130A_F: 5’ AGAAATGTTTGGAGAATTCGCAACTGGGAAGACCCAGATC 3’) | Custom order: Integrated DNA Technologies (IDT) | N/A |
| II3A mutagenesis primers (R130A_R: 5’ GATCTGGGTCTTCCCAGTTGCGAATTCTCCAAACATTTCT 3’) | Custom order: Integrated DNA Technologies (IDT) | N/A |
| II3A mutagenesis primers (R303A_F: 5’ AACAACCAGATTGTATCTGGCGAAAGGAAGAGGGGAAACC 3’) | Custom order: Integrated DNA Technologies (IDT) | N/A |
| II3A mutagenesis primers (R303A_R: 5’ GGTTTCCCCTCTTCCTTTCGCCAGATACAATCTGGTTGTT 3’) | Custom order: Integrated DNA Technologies (IDT) | N/A |
| II3A mutagenesis primers (K131A_F: 5’ GGGGAAACCAGAATCTGCGCAATCTACGACTCTCCCTG 3’) | Custom order: Integrated DNA Technologies (IDT) | N/A |
| II3A mutagenesis primers (K131A_R: 5’ CAGGGAGAGTCGTAGATTGCGCAGATTCTGGTTTCCCC 3’) | Custom order: Integrated DNA Technologies (IDT) | N/A |
| RT-qPCR primer against RAD51 F: TCTCTGGCAGTGATGTCCTGGA R: TAAAGGGCGGTGGCACTGTCTA |
Custom order: Integrated DNA Technologies (IDT) | N/A |
| RT-qPCR primer against GAPDH F: CTGTTGCTGTAGCCAAATTCGT R: ACCCACTCCTCCACCTTTGAC |
Custom order: Integrated DNA Technologies (IDT) | N/A |
| Software and algorithms | ||
| Trimmomatic v0.39 | Bolger et al.51 | RRID:SCR_011848 |
| FASTQC v0.11.5 | Babraham Bioinformatics | RRID:SCR_014583 |
| BWA-ALN | Li52 | RRID:SCR_010910 |
| SAMtools v1.8 | Li et al.53 | RRID:SCR_002105 |
| deepTools v3.5.1 | Ramírez et al.54 | RRID:SCR_016366 |
| GraphPAD Prism v8.4.3 | N/A | RRID:SCR_002798 |
| Python v3.8.8 | Van Rossum and Drake Jr55 | RRID:SCR_008394 |
| Matplotplib v3.4.2 | Hunter56 | RRID:SCR_008624 |
| NumPy v1.19.1 | Harris et al.57 | RRID:SCR_008633 |
| Pandas v1.2.3 | McKinney58 | RRID:SCR_018214 |
| pyGenomeTracks v3.6 | Ramírez et al.59 Lopez-Delisle et al.60 |
N/A |
| Fiji v2.0.0 | Schindelin et al.63 | RRID:SCR_002285 |
| Seaborn v0.11.1 | Waskom64 | - |
| Olympus FV1000 Software FV10-ASW v4.2 | Olympus | RRID:SCR_014215 |
| Cytoflex LX CytExpert v2.3.0.84 | Cytoflex | RRID:SCR_017217 |
| FlowJo v10.6.2 | FlowJo | RRID:SCR_008520 |
| Q-Rex software | QIAGEN | RRID:SCR_015740 |
| Other | ||
| FISH probe against centromere HORs (Cy3) 5’ ATTCGTTGGAAACGGGA 3’ |
PNABio | Cat#F3009 |
| FISH probe against telomeres (Cy5) 5’ CCCTAACCCTAACCCTAA 3’ |
PNABio | Cat#F1003 |
| FISH probe against HSat2 (A488) 5’ TCGAGTCCATTCGATGAT 3’ |
Custom order from PNABio | N/A |
| FISH probe against HSat3 (A488) 5’ TCCACTCGGGTTGATT 3’ |
Custom order from PNABio | N/A |
| Publicly available NGS dataset: HCT116 GLOE-seq (Sriramachandran et al. 2020) | NCBI Sequence Read Archive | SRR9676440 |
| Publicly available NGS dataset: HCT116 END-seq (Canela et al. 2019) | NCBI Sequence Read Archive | SRR8870099 |
| Publicly available NGS dataset: HCT116 input (ENCODE) | NCBI Sequence Read Archive | SRR577511 |
| Publicly available NGS dataset: CHM13 CUT&RUN CENP-A (Logsdon et al. 2021) | NCBI Sequence Read Archive | SRR15395852 |
| Publicly available NGS dataset: CHM13 CUT&RUN IgG (Logsdon et al. 2021) | NCBI Sequence Read Archive | SRR15395848 |
| Publicly available NGS dataset: CHM13 ChIP-seq CENP-A (Logsdon et al. 2021) | NCBI Sequence Read Archive | SRR13278683 |
| Publicly available NGS dataset: CHM13 ChIP-seq input (Logsdon et al. 2021) | NCBI Sequence Read Archive | SRR13278681 |
| Publicly available NGS dataset: iNeuron END-seq (Wu et al. 2021) | NCBI Sequence Read Archive | SRR13764826 |
| Publicly available NGS dataset: iNeuron input (Wu et al. 2021) | NCBI Sequence Read Archive | SRR13764817 |
| Publicly available NGS dataset: MS4221 CENP-A ChIP-seq (Hasson et al. 2013) | NCBI Sequence Read Archive | SRR8870099 |
| Publicly available NGS dataset: MS4221 input ChIP-seq (Hasson et al. 2013) | NCBI Sequence Read Archive | SRR577511 |