Complete protease inhibitor edta

CUT&RUN (protocol V.3) was performed according to Meers et al. (2019) (link) with minor modifications. In brief, 500,000 OSCs were bound to 10 µL of Concanavalin A-coated magnetic beads (Polysciences 86057-3) and lysed using Dig wash buffer (20 mM HEPES at pH 7.3, 150 mM NaCl, 0.5 mM spermidine, 0.01% digitonin, Roche Complete protease inhibitor EDTA). Bead-bound cells were incubated with 0.5 µg of respective antibody (Supplemental Table S7) at 4°C overnight on a nutator. Afterward, cells were washed twice, resuspended in Dig wash buffer containing 700 ng/mL pAG-MNase (produced in house), and incubated for 1 h at 4°C on a nutator. Cells were washed twice and resuspended in Dig-wash buffer containing 2 mM Ca²⁺ to activate pAG-MNase. Reaction was stopped by the addition of 2× STOP buffer, and samples were incubated at 500 rpm for 15 min at 37°C mixing to release DNA fragments into solution. After centrifugation, 0.1% SDS and 0.2 μg/μL Proteinase K were added to the supernatant and samples were incubated for 1 h at 55°C. DNA was purified using a DNA purification kit and libraries were prepared following the manufacturer's instructions with NEBNext Ultra II DNA library preparation kit for Illumina. Sequencing was performed on a HiSeqV4 using 50-bp single-end mode.

Polysome profiling was conducted following the protocol described in a previous study (22 (link)). In detail, one 10 cm dish of A549 cells was treated with 100 μg/ml CHX in DMEM at 37°C for 5 min prior to harvesting, and the cells were then washed twice with cold PBS containing 100 μg/ml CHX. Samples were lysed with 800 μl of lysis buffer containing 10 mM Tris–HCl (pH 7.4), 5 mM MgCl₂, 100 mM KCl, 1% Triton X-100, 2 mM DTT, 100 μg/ml CHX, Complete Protease Inhibitor EDTA-free (Roche, 4693132001) and 20 U/mL SUPERase-In RNase Inhibitor and were then harvested by scraping, transferred to Eppendorf tubes, and incubated on ice for 10 min. Lysates were centrifuged at 10 000 × g for 10 min at 4°C. RNA concentrations were measured with a Nanodrop UV spectrophotometer (Thermo Fisher Scientific). Equal amounts of lysates were layered onto a linear sucrose gradient (10–50%, w/v) with gradient buffer containing 20 mM HEPES–KOH (pH 7.4), 5 mM MgCl₂, 100 mM KCl, 2 mM DTT, 100 μg/ml CHX, and 20 U/ml SUPERase-In RNase Inhibitor, and were then centrifuged in an SW41 Ti rotor (Beckman) for 2 h at 36 000 rpm at 4°C. Samples were fractionated and analyzed with a Gradient Station (BioComp).