During gradient elution, fractions of ∼300 µl were collected every 14 s. For RNA isolation, 300 µl urea buffer (10 mM Tris, pH 7.5, 350 mM NaCl, 10 mM EDTA, 1% SDS, and 7 M urea) containing 25 fmol rabbit HBB2 in vitro transcript and 300 µl phenol:chloroform:isamylalcohol (25:24:1) were added to each fraction. After phase separation, RNA was isolated from the aqueous phase and precipitated using isopropanol. RNA levels in the different fractions were subsequently analyzed by quantitative PCR (qPCR) as follows: RNA was reverse transcribed using Moloney murine leukemia virus reverse transcription (Promega), followed by cDNA amplification using the PowerUp SYBR Green Master Mix (Thermo Fisher Scientific) and the QuantStudio 5 Real-TimePCR system (Thermo Fisher Scientific). All threshold count values were normalized to the HBB2 spike-in transcript to correct for isolation differences.
For protein purification, 300 µl Tris-HCl (20 mM, pH 7.5) and 10 µl StrataClear beads were added to each fraction. Samples were rotated end-over-end at 4°C overnight and centrifuged at ∼100 g for 2 min, and proteins were eluted from the beads using SDS sample buffer.
For protein purification, 300 µl Tris-HCl (20 mM, pH 7.5) and 10 µl StrataClear beads were added to each fraction. Samples were rotated end-over-end at 4°C overnight and centrifuged at ∼100 g for 2 min, and proteins were eluted from the beads using SDS sample buffer.