P8920

For prometaphase samples, washed mitotic cells were immediately prepared for downstream analysis. Remaining samples were re-cultured in standard media for synchronous release into G1 and collected at indicated times. For early time points, both floating and adherent re-cultured cells were collected for analysis. After 5 hours release from nocodazole, only adherent cells were collected.
Approximately 5 × 10⁶ cells at each time point were fixed in 1% Formaldehyde (Fisher BP531-25) diluted in serum-free DMEM for Hi-C analysis as described in Belaghzal et al. ^{49 (link)}. For cell cycle analysis, approximately 1 × 10⁶ cells at each time point were fixed in 86% cold ethanol (Fisher 04-355-222) and stored at −20°C. For chromatin association protein analysis, approximately 5 × 10⁶ cells at each time point were pelleted, flash frozen, and stored at −80°C. Additional samples were collected for fluorescent microscopy. Floating mitotic cells were resuspended in 1.5 mL 4% PFA (EMS 15710) (diluted in 1× PBS), transferred onto a Poly-L-lysine-coated coverslip (Sigma P8920) in a 6 well plate, and spun at 1500xg for 15 min. Cells adherent to coverslips at later time points were fixed in 4% PFA for 15 minutes at 20°C. All coverslips were washed 3× in 1× PBS and stored in 1× PBS at 4°C.

We prepared two kinds of bead samples in this manuscript: fluorescent bead layers for measuring PSFs at the coverslip surface and fluorescent beads embedded in 3D scattering gels for measuring PSF degradation at depth. Fluorescent bead layers were prepared by coating 24 × 50 mm #1.5 coverslips (VWR, 48393241) with 100 mg/ml poly-L lysine (Sigma, P8920), depositing 20–40 μL of 100 nm diameter yellow–green fluorescent beads (Invitrogen, F8803, 1:1000 dilution in water) on the coverslips, waiting for 10 min, and gently washing the coverslips in water to remove excess beads from the coverslip. Samples were then immersed in water for imaging. Fluorescent beads in 3D scattering gel samples were prepared by suspending 100 nm diameter yellow–green fluorescent beads at 1:250 dilution into a solution containing 5% agarose (Sigma, A9539) and 2.5% nonfluorescent 0.062 μm scattering polystyrene beads (Bangs Laboratories, PS02N). This mixture was vortexed vigorously and sonicated for 2 min before heating. After heating, the gelled mixture was deposited on a #1.5 coverglass bottomed dish (Matek, P35G-1.5-14-C), allowed to cool, and then immersed in water for imaging.

Single-cell suspensions were spun onto glass slides using a cytocentrifuge
(Shandon) or were grown on polylysine-coated slides (p8920, Sigma), as described^{13 (link)}. Cells were fixed at room temperature in 2%
paraformaldehyde for 20 min, permeabilized in TBS-Tween 20 for 20 min, washed three times
in PBS, and then blocked with 5% bovine serum albumin in PBS for 2 h before addition of
primary antibodies against BCL9 (ab37305, Abcam, 1:200), β-catenin (CAT5-H10,
Zymed, 1:100), Myeloperoxidase (A0398, DAKO, 1:100), CyPA (ERPR7511, Abcam, 1:200), Alexa
Fluor 647-conjugated CD147 (HIM6, Biolegend, 1:200), or FITC-conjugated CD138 (MI15,
Becton Dickinson, 1:200). Cells were incubated overnight with primary antibodies at 4
°C, and then washed three times in PBS before staining with secondary antibodies
conjugated to Alexa Fluor 488 (A11034, Molecular Probes, 1:200) or Alexa Fluor 546
(A11035, Molecular Probes, 1:200). Images were acquired with the aid of a Bio-Rad Radiance
2000 laser scanning confocal or Nikon Eclipse E800 phase-contrast microscope. Optimal
antibody concentrations were used according to manufacturer's recomendations.

HEK293T cells (ATCC; CRL-11268) were grown and split following standard protocols as described previously.^{37 (link)} HEK cells at low-passage-number (< 10 passages) were plated at a confluence of 30% onto 10-cm dishes coated with gelatin (Stemcell Technologies; 07903) or 14 mm glass bottom dishes (CellVis, D35-14-1.5-N) coated with 40 μg/ml poly-L-lysine-coated (P8920, Sigma-Aldrich). Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and penicillin/streptomycin was used as the culture medium. Cells were grown at 37 °C and 5% CO₂.
When cells reached 50-70% confluence, genes were delivered by either lentivirus or TransIT-293 (Mirus; MIR2700) transfection kit. In lentiviral transduction, the high-titer lentiviral vectors were first pre-mixed at the designated ratio and diluted to 10% of the initial concentration by DMEM medium. The cells’ culture medium was then replaced by the fresh lentivirus-containing DMEM medium, and the cells were further incubated at 37 °C and 5% CO₂ for ~24 h. In the TransIT-293 transfection, 1 μg plasmids at the designated ratio were first diluted by 100 μl Opti-MEM medium, followed by the addition of 3 μl TransIT-293 reagent. The cocktail was incubated at room temperature for 15 min and diluted 10-fold into DMEM on the culture dishes. The cells were then further incubated at 37 °C and 5% CO₂ for ~24 h.