G8772

Frozen pellets were re-suspended in 250 µL of 65 °C sterilized bead buffer (7.5 mM NH₄OAc; 10 mM EDTA, pH 8.0) and then transferred to 1.5 mL tubes prewarmed at 65 °C acid-washed glass beads (G8772-100 Merck) suspended in 25 µL of 10% filtered SDS and 300 µL of acid phenol chloroform pH 4.5. The tubes were vortexed for 1 min then incubated at 65 °C for 1 min. This was repeated three times. After heating the tubes at 65 °C for 10 min, and after an additional 1 min-round of vortex, the samples were centrifuged at 16,000 × g, 25 °C, for 15 min. The supernatant was then transferred to a new tube with an equal volume of chloroform on ice. After vortexing the tubes, the tubes were centrifuged at 16,000 × g, 4 °C, for 15 min. The supernatant was transferred to a new tube with 7.5 M NH₄OAc (final concentration of 0.51 M). The tubes were then vortexed. After adding 100% cold ethanol, the tubes were put at –80 °C. The samples were then centrifuged at 20,400 × g at 4 °C for 30 min. After discarding the supernatant, the pellet was washed in 75% ice-cold ethanol was then added. After a final centrifugation at 20,400 × g, 4 °C, for 15 min, the supernatant was discarded. The pellet was dried using a vacuum-centrifuge for 5 min and re-suspended on ice with cold RNAse-free water and stored at –80 °C. RNA quality and concentration was measured using a Nanodrop spectrophotometer (ND-1000).

A P2 virus encoding RBBP6_UBL-SII and a P2 virus carrying a gene of one of the CPSF73 variants (CPSF73_FL, CPSF73_NTD, or CPSF73_CTD) were used to coinfect Sf9 cells at ∼2 million cells/mL. The cultures were harvested after 3 d by centrifugation and washed in ice-cold PBS. The cell pellets were lysed using glass beads (Merck G8772) in lysis buffer [50 mM HEPES-NaOH at pH 8.0, 300 mM NaCl, 1 mM TCEP, 2 mM Mg(OAc)₂] supplemented with two protease inhibitor tablets per 50 mL of buffer. The lysates were cleared by centrifugation and applied to Strep-Tactin beads. After a 2-h incubation, the beads were washed in lysis buffer, and the bound proteins were eluted by incubating the samples in NuPAGE LDS sample buffer (Invitrogen NP0007) for 2 min at 98°C. The eluted proteins were analyzed on a NuPAGE 4%–12% Bis-Tris 1.0-mm mini protein gel (Invitrogen NP0321), and the gel was stained with Instant Blue (Abcam 119211).

Cells were washed with PBS and the pellet resuspended in lysis buffer (80% methanol + 5% Trifluoroacetic acid) spiked with 2.5 μM 1,7-diaminoheptane (Sigma). The cell suspension, together with acid-washed glass beads (G8772, Sigma), was transferred to a bead beater tube and homogenized in a bead beater (Precellys 24, Bertin Technologies) for four cycles (6500 Hz, 45 s) with 1 minute of ice incubation between each cycle. The homogenized samples were centrifuged at 13,000 g for 20 minutes at 4°C. The supernatant was collected and dried overnight. For chemical derivatization, 200 μL trifluoroacetic anhydride was added to the dried pellet and incubated at 60°C for 1 hour, shaking at 1200 rpm. The derivatization product was dried, re-suspended in 30 μL isopropanol and transferred to glass loading-vials. The samples were analyzed using a GCxGC-MS system as described (Yu et al., 2017 (link)). The following parameters were used for quantification of the 1D-GC-qMS data: type: area, slope: 1000/min, width: 0.04 s, drift 0/min and T. DBL: 1000 min without any smoothing methods used. Cellular spermidine amount was normalized to total protein levels determined by BCA assay for each sample first followed by comparison between samples.