Microsome and MBP isolation

The isolation of microsomes and MBPs for small RNA sequencing was performed as described (Li et al., 2013). In brief, 2 g of seedlings were ground in 7 ml microsome isolation buffer (MEB: 100 mM Tris-HCl, pH7.5, 5 mM EGTA, 15 mM MgCl₂, 5 mM DTT, 0.3M sucrose, 50 U/ml superaseIN (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), 50 μg/ml cycloheximide, 50 μg/ml chloramphenicol, and a complete proteinase inhibitor cocktail (Roche)), and the cell lysate was filtered by 2 layers of miracloth and centrifuged at 10,000 g twice to remove debris. A 100 µl aliquot was taken from the clarified supernatant as input, and the rest was applied onto a sucrose step gradient (2.5 ml 20%/2.5 ml 60% sucrose), and centrifuged at 100,000 g in an SW41 rotor (Beckman coulter) for 1 hr. Microsomes were recovered from the interface of the two sucrose layers, diluted with 10 volumes of MEB and precipitated by centrifugation at 100,000 g for 30 min.

To isolate MBP, the microsome preparation was lysed with 8 ml polysome isolation buffer (0.2M Tris-HCl, pH9.0, 0.2M KCl, 0.025M EGTA, 0.035M MgCl2, 0.2% Brij-35, 0.2% Triton X-100, 0.2% Igepal CA630, 0.2% Tween 20, 0.2% polyoxyethylene 10 tridecyl ether, 5 mM DTT, 1 mM PMSF, 50 μg/ml cycloheximide, 50 μg/ml chloramphenicol and 2.5 U/ml superaseIN). The lysate was kept on ice for 30 min, and then loaded on the top of a sucrose cushion (0.4M Tris-HCL, pH9.0, 0.2M KCl, 0.005M EGTA, 0.035m MgCl₂, 1.75M sucrose, 5 mM DTT, 50 μg/ml cycloheximide, and 50 μg/ml chloramphenicol) and centrifuged at 170,000 g for 3 hr. The pellet was dissolved with resuspension buffer (0.2M Tris-HCl, pH9.0, 0.2M KCl, 0.025M EGTA, 0.035M MgCl₂, 5 mM DTT, 50 μg/ml cycloheximide, and 50 μg/ml chloramphenicol) and the solution was transferred into a new tube as the MBP fraction. An aliquot of the MBP was subjected to 15–50% sucrose gradient centrifugation for polysome integrity evaluation, and the rest was used for sRNA and mRNA sequencing.