Extended Data Fig. 2: Optimization of P1 nuclease digestion and cell lysis conditions.

a, Polysome collapse efficiency analysis comparing lysis with either pH 6.5 or pH 7.5 polysome lysis buffer and addition of a range of P1 nuclease U/µg. All assays were carried out with lysate measuring 30 µg of total RNA diluted to 200 µL with either pH 6.5 or pH 7.5 polysome buffer. At the point of nuclease digestion, pH 7.5 lysate was adjusted to ~pH 6.5 with 14 µl 300 mM Bis-Tris (pH 6.0) and pH 6.5 lysate was supplemented with an additional 14 µL of pH 6.5 polysome buffer. Collapse efficiency was calculated as the ratio of the integrated monosome peak absorbance relative to the integrated polysome region absorbance, normalized by the undigested control (n = 1 for each condition, except n = 2 for pH 6.5 with 3.33 U/µg or 20 U/µg and pH 7.5 with 0 U/µg or 10 U/µg). b, Comparison of polysome collapse efficiency by P1 nuclease digestion at either 30 °C or 37 °C using Calu-3 human cell lysate. Briefly, lysate measuring 15 µg of total RNA was diluted to 200 µL in pH 7.5 polysome buffer and pH adjusted with 14 µL of 300 mM Bis-Tris (pH 6.0) before supplemented with P1 nuclease and digestion at 30 °C or 37 °C. Undigested control incubated at 4 °C for an hour without nuclease (n = 2 for 4 °C no-nuclease controls, n = 2 or 3 for 15 U/µg as shown, and n = 1 for 20 U/µg). c, Representative polysome profile from P1 nuclease digestion of Calu-3 human cell lysate at 30 °C or 37 °C with nuclease at 15 U/µg total RNA. Undigested control incubated at 4 °C for an hour without nuclease.