GoKawiilCell culture
HCT116 cells and derivative cell lines (Supplementary Table 1) were grown in DMEM (Life Technologies, 41966-029) supplemented with 10% fetal bovine serum (FBS, GIBCO), 100 U ml−1 penicillin and 100 mg ml−1 streptomycin in an atmosphere containing 6% CO 2 at 37 °C. HCT116 OsTIR (F74G), mAID-mClover-RAD21 HCT116 OsTIR (F74G)6, WAPL-mClover-mAID HCT116 OsTIR (F74G)4 and CTCF-mClover-mAID HCT116 OsTIR (F74G)6 were provided by M. Kanemaki. None of the cell lines were authenticated in house. All cell lines used in this study were subjected to regular mycoplasma testing in house, and all of them consistently reported negative results. To degrade mAID-tagged proteins, cells were treated with 2 µM 5-Ph-IAA (aux; BioAcademia). To degrade Halo-SMARCAL1, cells were treated with Halo-PROTAC3 at 1:1,000 for the indicated times.
Cell line generation
Cell lines were generated following published protocols56,57. HCT116 OsTIR (F74G) cells were transfected with CRISPR-Cas9 and donor plasmids using FuGENE HD Transfection Reagent (Promega) in a 12-well plate following the instructions of the manufacturer. Two days after transfection, cells were plated in 10 cm2 dishes and selected with antibiotics. Selected clones were isolated and confirmed for protein expression of the modified alleles. Oligonucleotides to generate sgRNA or amplify homology arms to build donor plasmids are provided in Supplementary Table 1.
RNA interference
For RNA interference, cells were transfected with the indicated siRNAs (Supplementary Table 1) for the indicated times and concentrations. Transfections were carried out using RNAiMax (Thermo Fisher) according to the instructions of the manufacturer.
Lentiviral transduction
For cell lines expressing SMC1WT, SMC13D or SMC14E, SMC1-3×Flag variant genes were cloned into LT3GEPIR (generated by GeneScript) and transfected into to HEK293T cells together with the lentiviral packaging vectors. After 48 h, lentiviral culture medium was harvested, filtered and added to SMC1-mClover-mAID HCT116 OsTIR (F74G) cells. After infection for 24 h, puromycin (2 μg ml−1, InvivoGen) resistant single cells were isolated. To induce the expression of the SMC1 variants, cells were treated with 1 µg ml−1 dox (Sigma-Aldrich).
iPOND–MS
iPOND was performed as described33,58 with minor modifications. HCT116 cells were labelled with 10 µM EdU for 10 min and treated with the different drugs as indicated in Extended Data Fig. 1a. Cells were crosslinked with 1% formaldehyde for 20 min at room temperature (RT), quenched with 0.125 M glycine for 5 min, and washed three times with cold PBS. EdU was linked to biotin, after permeabilization with 0.25% Triton X-100/PBS for 30 min, by incubating in click reaction buffer (10 mM sodium-l-ascorbate, 20 µM biotin azide (Vanderbilt University) and 2 mM CuSO 4 ) at RT for 1 h on a rotator. Cells were washed twice with PBS, resuspended in lysis buffer (50 mM Tris-HCl, pH 8.0 and 1% SDS) supplemented with protease inhibitors, and chromatin was solubilized by sonication in a Bioruptor Pico (Diagenode) at 4 °C 10 min (30 s on and 30 s off cycles). After centrifugation for 10 min at 16,000g, supernatants were diluted with 1:1 PBS (vol/vol) containing protease inhibitors and incubated overnight with myOne streptavidin C1 dynabeads (Thermo Fisher). Beads were washed once with lysis buffer, once with 1 M NaCl, twice with lysis buffer and once with PBS. Captured proteins were digested on beads using 500 ng of sequencing grade modified trypsin (Promega, V5111), including reduction and alkylation of cysteines with tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and 2-chloroacetamide (ClAA) addition, respectively. Resulting peptides were cleaned using the Phoenix kit (Preomics) according to instructions in the kit. Liquid chromatography-tandem mass spectrometry analysis of peptide mixture was conducted on an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher) directly coupled to an ACQUITY UPLC M-Class System (Waters) configured for 75-μm scale single-pump trapping. Peptides were separated on a nanoEase HSS C18 T3, 100 A, 75 μm × 250 mm analytical column (Waters, PN: 186008818) at a constant flow rate of 300 nl min−1 applying a peace-wise linear gradient from 5% to 33% solvent B in 45 min (solvent A: water, including 0.1% formic acid; solvent B: acetonitrile, including 0.1% formic acid). MS data acquisition was conducted in data-independent mode. DIA scans were acquired in the Orbitrap mass analyser at 15,000 Resolution (normalized AGC target: 3,000%, maxIT: 23 ms) covering the m/z range from 350 to 1,050 in 70 non-overlapping isolation windows. Precursors were quadrupole isolated at 10m/z and HCD fragmented at an NCE of 28 (calculated for the middle of the isolation window and charge 2).
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