Streptavidin sepharose bead

ChIRP was performed as previously with some modifications⁷⁶ . Antisense DNA probes which were separated into two groups (even and odd) were designed against the full-length MAS sequence and biotinylated at the 3’ end (Invitrogen). A set of probes against lacZ RNA was used as negative control.
Col-0 seedlings (1 g) were crosslinked in 1% (vol/vol) formaldehyde (Sigma-Aldrich) at room temperature for 20 min in a vacuum. Crosslinking was quenched with 0.125 M glycine for 5 min. Nuclei were isolated as described in the NRO assay and were sonicated. Chromatin was diluted in 2 volumes of hybridization buffer (750 mM NaCl, 1% SDS, 50 mM Tris-HCl pH 7.0, 1 mM EDTA, 15% formamide, 0.1 mM PMSF, 1 × protease inhibitor, and 350 U/mL RNase inhibitor) and was mixed gently. After preclearance with Streptavidin Sepharose beads (GE Healthcare), 100 pmol of probes were added and mixed by end-to-end rotation at 37 °C for 4 h. Washed Streptavidin Sepharose beads (30 μL) were added, and the reaction was performed at 37 °C for 30 min with rotation. Then beads were washed two times with high-salt wash buffer (2 × SSC, 0.5% SDS, 1 mM DTT, and 1 mM PMSF) and two times with low-salt wash buffer (0.1 × SSC, 0.5% SDS, 1 mM DTT, and 1 mM PMSF) for 5 min each time at room temperature. DNA and RNA were purified and analyzed by qPCR. Probes and primer sequences are provided in Supplementary Data 9.

NLS1–HK2 cDNA was cloned in-frame with BirA* fused to NLS1 into a tetracycline-inducible pcDNA5 FLP recombinase target/tetracycline operator (FRT/TO) expression vector, which was then transfected into Flp-In T-REx HEK293 cells. The cells were collected and pelleted (800g for 3 min), the pellet was washed twice with PBS and the dried pellets were snap frozen. The pellets were lysed in 10 ml of modified RIPA lysis buffer at 4 °C for 1 h and then sonicated (30 s at 35% power; Sonic Dismembrator 500, Fisher Scientific) to disrupt the visible aggregates. The lysate was centrifuged at 35,000g for 30 min. The clarified supernatants were incubated with 30 l packed, pre-equilibrated streptavidin-Sepharose beads (GE) at 4 °C for 3 h on an end-over-end rotator. The beads were collected (800 g for 2 min) and washed six times with 50 mM ammonium bicarbonate (pH 8.3). The beads were then treated with l-1-tosylamide-2-phenylethyl chloromethyl ketone–trypsin (Promega) for 16 h at 37 °C on an end-over-end rotator. After 16 h, another 1 µl of l-1-tosylamide-2-phenylethyl chloromethyl ketone–trypsin was added and the sample was incubated in a water bath at 37 °C for 2 h. The supernatants were lyophilized and stored at 4 °C for downstream mass spectrometry analysis. Two biological and two technical replicates were completed and the NLS1-HK2 interactors were normalized to the BirA* spectral counts.

HEK293T cells were transfected with pMCB-SBP-Flag-ECHS1 or pMCB-SBP-Flag, each containing a puromycin-resistance selection marker. The stable cell lines were established by selecting with 5 μM puromycin. The ECHS1 stable cells were lysed on ice with 0.1% NP-40 buffer containing protease inhibitors cocktail for 30 min. Cell lysate was incubated with Flag beads for 3 h followed by elution with Flag peptide. The resulted solution was incubated with streptavidin-Sepharose beads (GE Healthcare) for 3 h at 4 °C, and the beads were washed three times with NP-40 buffer followed by three times with 50 mM NH₄HCO_3.On-beads tryptic digestion were carried out at 37 °C overnight. The resulted peptides in supernatant were dried in Concentrator Plus (Eppendorf) before subject to mass spectrometry analysis.