GSM6685066: RNA bound with xPAC; Spodoptera frugiperda; OTHER
(1) The xPAC was expressed in Sf9 and the cells were centrifuged at 800 × g for 15 min and suspended in lysis buffer (25 mM Tris, 150 mM NaCl, pH 8.0) with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 0.8 µM aprotinin, 2 µM pepstatin A and 5 µg/mL leupeptin). The collected cells were flash-frozen in liquid nitrogen and stored at −80 °C before purification. The thawed cells were solubilized by adding 2% (w/v) n-Dodecyl β-D-maltoside (DDM, Anatrace) and stirring at 4 °C for 2 h. The resulting cell lysate was centrifuged at 27,000 × g at 4 °C for 30 min. The supernatant was incubated with Strep-tactin resin (GE Healthcare) at 4 °C for 1 h. The resin was washed with lysis buffer supplemented with either 0.06% (w/v) digitonin (Biosynth). Proteins were eluted with elution buffer (25 mM Tris, 150 mM NaCl, 5 mM desthiobiotin, pH 8.0) containing with 0.06 % digitonin. The protein samples were further purified by size-exclusion chromatography (SEC, Superose 6 increase 10/300 GL, GE Healthcare) in lysis buffer containing 0.06% digitonin. The purified xPAC proteins were used to extract RNA by using a phenol-chloroform extraction followed by ethanol precipitation. (2) The hPAC was expressed in HEK293F cells. The purification procedure was similar to that described for xPAC, with the following modification. The wash buffer contained 25 mM Tris pH 8.0, 150 mM NaCl, and 0.05% DDM. The protein was eluted with elution buffer containing 25 mM Tris, pH 8.0, 150 mM NaCl, 0.05% DDM, and 5 mM desthiobiotin. The RNAs enriched in the hPAC samples were extracted using the phenol-chloroform method followed by ethanol precipitation. The purified RNA was demethylated by rtStarTM tRF&tiRNA Pretreatment Kit (Arraystar, AS-FS-005) and partially hydrolyzed according to Hydro-tRNAseq mothod. The library was prepared by using NEBNext® Multiplex Small RNA Library Prep Set for Illumina® kit (New England Biolabs, E7300L/E7850L)