Key resources table.
| Reagent type (species) or resource |
Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | U2OS | (Hansen et al., 2017) | U2OS | Wild-type U2OS cell line. RRID:CVCL_0042 |
| Cell line (Mus musculus) | mESC | (Pettitt et al., 2009) and UC Davis KOMP Repository | JM8.N4 mESC |
https://www.komp.org/pdf.php?cloneID=8669
(RRID:CVCL_J962) |
| Cell line (Mus musculus) | mESC C59 Halo-CTCF | (Hansen et al., 2017) | mESC C59 Halo-mCTCF | mESC (JM8.N4) endogenous knock-in cell line where both endogenous copies of Ctcf have been N-terminally tagged with FLAG-HaloTag and where both endogenous copies of Rad21 have been C-terminally tagged with SNAPf-V5. Clone 59 |
| Cell line (Mus musculus) | mESC C87 Halo-CTCF | (Hansen et al., 2017) | mESC C87 Halo-mCTCF | mESC (JM8.N4) endogenous knock-in cell line where both endogenous copies of Ctcf have been N-terminally tagged with FLAG-HaloTag. Clone 87 |
| Cell line (Mus musculus) | mESC C45 Rad21-Halo | (Hansen et al., 2017) | mESC C45 mRad21-Halo | mESC (JM8.N4) endogenous knock-in cell line where both endogenous copies of Rad21 have been C-terminally tagged with HaloTag-V5. Clone 45. |
| Cell line (Mus musculus) |
mESC C3 Halo-Sox2 | (Teves et al., 2016) | mESC C3 Halo-Sox2 | mESC (JM8.N4) endogenous knock-in cell line where both endogenous copies of Sox2 have been N-terminally tagged with FLAG-HaloTag. Clone 3 |
| Cell line (Homo sapiens) | U2OS C32 Halo-CTCF | (Hansen et al., 2017) | U2OS C32 Halo-hCTCF | U2OS endogenous knock-in cell line where all endogenous copies of Ctcf have been N-terminally tagged with FLAG-HaloTag. Clone 32 |
| Cell line (Mus musculus) | mESC C85 Rad21-Halo-V5 het | This Paper | C85 | mESC (JM8.N4) endogenous knock-in cell line where one endogenous Rad21 allele is Halo-V5 tagged while the other allele is ‘near wild type’ (see Materials and methods). Clone 85 |
| Cell line (Mus musculus) | mESC B4 Rad21-Halo-V5/Rad21-SNAPf-3xFLAG | This Paper | B4 | mESC (JM8.N4) endogenous knock-in cell line where one endogenous Rad21 allele is Halo-V5 tagged while the other allele is SNAPf-3xFLAG tagged. Clone B4 |
| Cell line (Mus musculus) | mESC A2 Rad21-Halo-V5/Rad21-SNAPf-3xFLAG | This Paper | A2 | mESC (JM8.N4) endogenous knock-in cell line where one endogenous Rad21 allele is Halo-V5 tagged while the other allele is SNAPf-3xFLAG tagged. Clone A2 |
| Antibody | rabbit polyclonal anti-V5 |
Abcam | Cat. # ab9116, RRID:AB_307024 | (1:2000) for western blot (WB) |
| Antibody | mouse monoclonal mouse anti-V5 | ThermoFisher Scietific | Cat. # R960-25, RRID:AB_2556564; | (1:5000) for WB |
| Antibody | rabbit polyclonal anti-FLAG |
Sigma-Aldrich | Cat. # F7425, RRID:AB_439687 | (1:1000) for WB |
| Antibody | mouse monoclonal anti-FLAG |
Sigma-Aldrich | Cat. # F3165, RRID:AB_259529 | (1:5000) for WB |
| Antibody | rabbit polyclonal anti-Rad21 |
Abcam | Cat. # ab154769, RRID:AB_2783833 |
(1:2000) for WB |
| Antibody | mouse anti-Rad21 | Millipore | Cat. # 05–908, RRID:AB_417383 |
(1:5000) for WB |
| Antibody | mouse monoclonal anti-Halo |
Promega | Cat. # G9211, RRID:AB_2688011 |
(1:1000) for WB |
| Antibody | mouse monoclonal anti-βactin |
Sigma-Aldrich | Cat. # A2228, RRID:AB_476697 | (1:4000) for WB |
| Peptide, recombinant protein | 3xFLAG-Halo-CTCF-His6 | This Paper | 3xFLAG-Halo-CTCF-His6 | See Materials and methods. |
| Peptide, recombinant protein | His6-Rad21-Halo-3xFLAG | This paper | His6-Rad21-Halo-3xFLAG | See Materials and methods. |
| Recombinant DNA reagent | pHTCHaloTag | This paper | pHTCHaloTag | For FCS-calibrated imaging experiments (referred to as pHTCHaloTag), a stop codon was introduced into the pHTC HaloTag CMV-neo vector (Promega, #9PIG771) |
| Software, algorithm | Matlab | The Mathworks | MATLAB 2014b | https://www.mathworks.com/products/matlab.html |
| Software, algorithm | Flow cytometry analysis (Matlab) |
This paper | Flow cytometry analysis |
https://gitlab.com/tjian-darzacq-lab/cattoglio_et_al_absoluteabundance_2019 |
| Software, algorithm | FCSREAD (Matlab) | Mathworks File Exchange | FCSREAD (Matlab) |
https://www.mathworks.com/matlabcentral/fileexchange/8430-flow-cytometry-data-reader-and-visualization |
| Commercial assay, kit | Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit |
ThermoFisher Scientific | Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit | Cat. # C10425 |
| Chemical compound, drug | DAPI | Sigma-Aldrich | 4′,6-Diamidine-2′-phenylindole dihydrochloride | Cat. # 10236276001 |
| Chemical compound, drug | Halo-TMR | Promega | HaloTag TMR ligand | Cat. # G8251 |
| Chemical compound, drug | Halo-JF646 | (Grimm et al., 2015) | Halo-JF646 | Please contact Luke D Lavis for distribution. |
| Chemical compound, drug | Hoechst 33342 | Sigma-Aldrich | Hoechst 33342 | Cat. # B2261 |