Am2696

Ribosome display was performed using a custom PURExpress kit from New England Biolabs (NEB) that lacks release factors 1, 2 and 3, and T7 RNA polymerase. Specifically, we prepared a 200 µl master mix containing 80 µl of solution A, 60 µl of solution B, 4 µl of disulfide enhancers 1 and 2 (E6820S; NEB) (if required) and 4 µl of Superase In (AM2696; Thermo Fisher). We then injected the master mix into each lane of the flow cell, being careful to avoid the introduction of bubbles, before incubating the flow cell at 37 °C for 60 minutes. Once the incubation period was complete, we cooled the flow cell down to 20 °C, before washing and stabilizing the ribosomes with ribosome display buffer (50 mM Tris(hydroxymethyl)aminomethane acetate, 150 mM NaCl, 50 mM magnesium acetate, 0.1% Tween 20 and 1 U ml⁻¹ of Superase In (AM2696; Thermo Fisher), pH 7.5).
With the ribosomes stabilized by the display buffer, we block the flow cell with binding buffer (ribosome display buffer with 0.1% BSA; A9647; Sigma-Aldrich). After flow-cell blocking, we image the surface to determine a baseline for background fluorescence.

Nuclei were isolated using Nuclei EZ Lysis buffer (NUC-101; Sigma-Aldrich) supplemented with protease inhibitor (5892791001; Roche) and ribonuclease inhibitor (N2615, Promega and AM2696, Life Technologies).

For snATAC-seq, nuclei were isolated with Nuclei EZ Lysis buffer (NUC-101; Sigma-Aldrich) supplemented with protease inhibitor (5892791001; Roche). Samples were cut into <2 mm pieces, homogenized using a Dounce homogenizer (885302–0002; Kimble Chase) in 2 ml of ice-cold Nuclei EZ Lysis buffer, and incubated on ice for 5 min with an additional 2 ml of lysis buffer. The homogenate was filtered through a 40 μm cell strainer (43–50040–51; pluriSelect) and centrifuged at 500 × g for 5 min at 4 °C. The pellet was resuspended, washed with 4 ml of buffer, and incubated on ice for 5 min. Following centrifugation, the pellet was resuspended in Nuclei Buffer (10× Genomics, PN-2000153), filtered through a 5 μm cell strainer (43-50005-03, pluriSelect), and counted. For snRNA-seq preparation, the RNase inhibitors (Promega, N2615 and Life Technologies, AM2696) were added to the lysis buffer, and the pellet was ultimately resuspended in nuclei suspension buffer (1× PBS, 1% bovine serum albumin, 0.1% RNase inhibitor). Subsequently, 10X Chromium libraries were prepared according to manufacturer protocol.