FUS_PROTAC3.bio2.tech2
U2OS cells were cultured to 7080% confluence and treated with 2 M of G4L-PROTAC for 6 h before harvesting. To facilitate cellular uptake of the compound, 6 L of Endo-Porter (Endo-Porter PEG: 1 mM Endo-Porter in 10% polyethylene glycol, 1500 MW, aqueous) was added per 1 mL of culture medium together with G4L-PROTAC. Then the cells were detached using Accutase (Gibco, Cat. No. A1110501), quenched with full-growth DMEM, and centrifuged at 200  g for 5 minutes. The resulting cell pellets were fixed in 0.1% (w/v) formaldehyde in PBS for 2 minutes at room temperature and quenched with 110 mM glycine. Cells were then centrifuged at 300  g for 5 minutes at 4 C, washed, and resuspended in cold wash buffer (20 mM HEPES, pH 7.5, 150 mM KCl, 0.5 mM spermidine, cOmplete EDTA-free protease inhibitor cocktail, Roche, Cat. No. 11836170001). Cells were either used immediately or cryopreserved in wash buffer supplemented with 10% DMSO at 80 C. A final cell concentration of 6 million cells per ml was used for subsequent steps. Concanavalin A beads (15 l per sample; Bangs Laboratories, Cat. No. BP531) were washed twice with binding buffer (20 mM HEPES, pH 7.5, 10 mM KCl, 1 mM CaCl, 1 mM MnCl) and resuspended in 10 l of the same buffer. A 100 l suspension of cells was combined with 10 l of pre-washed beads in 0.2 ml PCR tubes and mixed on an Intelli Mixer RM-2M (ELMI) using mode C3 (cycle: 6 s at 108, 6 s at 252, 12 s pause) at 20 rpm for 10 minutes at room temperature. Bead-bound cells were gently washed twice with 100 l wash buffer using a magnetic stand and resuspended in 100 l of a 1:100 diluted 1st antibody solution. Control samples without 1st antibody were included. The incubations were carried out on the same mixer at 4  overnight. To remove unbound 1st antibodies, cells were washed three times with 200 l of Dig-wash buffer (wash buffer with 0.05% digitonin), followed by incubation with 100 l of guinea pig anti-rabbit IgG secondary antibody (antibodies-online, Cat. No. ABIN101961) diluted 1:100 in Dig-wash buffer for 1 hour at room temperature on the mixer. The pA-Tn5 adapter complex (2 M) was prepared as previously described and diluted 1:200 in Dig-300 buffer (20 mM HEPES, pH 7.5, 300 mM KCl, 0.5 mM spermidine, 0.01% digitonin, protease inhibitors)[i]. Cells were washed three times with 200 l Dig-wash buffer to remove excess antibodies before being incubated with 100 l of pA-Tn5 complex for 1 hour on the mixer at room temperature. Subsequently, cells were washed three times in 200 l Dig-300 buffer to eliminate unbound pA-Tn5 and resuspended in 200 l of tagmentation buffer (Dig-300 with 10 mM MgCl) for 1 hour at 37 C on the mixers. After tagmentation, cells were washed twice in 200 l TAPS buffer (10 mM TAPS, 0.2 mM EDTA), then resuspended in 100 l extraction buffer (10 mM Tris-HCl, pH 8.0, 0.5 mg/ml proteinase K [Thermo Scientific, Cat. No. EO0491], 0.5% SDS). Samples were incubated at 55 C for 1.5 hours at 800 rpm. DNA was purified using the DNA Clean & Concentrator-5 kit (Zymo Research, Cat. No. D4013) and eluted in 25 l elution buffer following a 10-minute incubation at room temperature. For library amplification, 21 l of tagmented DNA was combined with 2 l each of uniquely barcoded v2 Ad1.x and Ad2.x primers (10 M each)[ii], and 25 l of NEBNext Ultra II Q5 2 PCR Master Mix (New England Biolabs, Cat. No. M0544). PCR cycling conditions were: 72 C for 5 min; 98 C for 30 s; followed by 10 cycles of 98 C for 10 s and 63 C for 10 s; and a final extension at 72 C for 1 min. Libraries were purified with 1.3 volume (65 l) of Ampure XP beads (Beckman Coulter, Cat. No. A63882), incubated at room temperature for 10 min, and washed twice with 80% ethanol. DNA was eluted in 25 l of 10 mM Tris-HCl (pH 8.0). Library size distribution was verified using the TapeStation with High Sensitivity D1000 ScreenTape (Agilent, Cat. No. 5067-5584), and concentrations were quantified using the NEBNext Library Quant Kit (New England Biolabs, Cat. No. E7630L). Pooled libraries were size-selected by adding 0.4 volume Ampure XP beads, transferring the supernatant to a new tube, then adding 1.3 volume Ampure XP beads for final cleanup. Beads were washed twice with 80% ethanol and DNA was eluted in 30 l of 10 mM Tris-HCl, pH 8.0. Sequencing was performed on an Illumina NextSeq 2000 using the NextSeq 1000/2000 P2 reagent kit (100 cycles, Cat. No. 20046811). Experiments were carried out in two biological replicates with two technical replicates each.
SUB15306132
FUS_PROTAC3.bio2.tech2