Sodium dodecyl sulfate (sds)

Forty milliliters of hemolymph collected from 100 infected silkworms at 6 days post-inoculation were used for purification of polyhedra according to our previous report [35 (link)]. Briefly, after the hemolymph was centrifuged for 10 min at 6000× g, the obtained pellet was washed 3–5 times with 0.1% sodium dodecyl sulfate (SDS) (Sangon, Shanghai, China) until the pellet turned white. The pellet was washed with 1× PBS twice to obtain the purified polyhedra.

For ChIRP-MS, the ovarian cell suspension was reversibly cross-linked in situ with formaldehyde (Sangon Biotech, Shanghai, China), followed by cleaving with lysis buffer (50 mM Tris-HCl pH 7.0 (Sigma-Aldrich, St. Louis, MO, USA), 1% SDS (Sangon Biotech), 10 mM EDTA (Sigma-Aldrich), 1 mM PMSF (Sangon Biotech)) and hybridization with biotin-labeled RNA probes (control probe and Gm2044 probe). After the non-specific binding proteins were washed out under strong denaturation conditions, the obtained RNA-binding proteins were identified and quantitatively analyzed by liquid chromatography-tandem MS. Then, the interactive protein profile of lncRNA Gm2044 was constructed and analyzed with gene ontology (GO, http://geneontology.org/), the Kyoto Encyclopedia of Genes and Genomes (KEGG, https://www.genome.jp/kegg/) and STRING (https://cn.string-db.org/). The probes of lncRNA Gm2044 were listed in Table S3, and the control probe was described in a previous study [17 (link)]. For RNA IP, the corresponding RNA-protein complexes were precipitated using antibodies against EEF2 protein (ABclonal Technology, Wuhan, China), and the purified protein and RNA were then analyzed according to the protocol in previous studies [18 (link), 19 (link)].

RNA samples from the cells of different strains were extracted by using the Trizol^TM reagent according to the instructions (Thermo, US). As an optimization step, we noticed that the cell-broken efficiency was greatly enhanced by mixing the cells with 0.05% SDS (Sangon Biotech, China) and 20 mg/mL lysozyme (Solarbio, China) as the final concentrations for 20 min at 37 °C before treating with Trizol^TM. Total cDNA was synthesized using the All-In-One RT MasterMix (ABM). Residual DNA in the extracted RNA was further removed by using DNase I (Vazyme, China). Some of the gDNA-free RNA was saved for the detection of gDNA contamination.
RT-qPCR was performed with the BestarVR SybrGreen qPCR Mastermix (DBI) and the LightCycler 480II instrument (Roche) according to the specification. For calculation of relative expression levels of the target genes, gyrB was used as the internal standard, and the quantification method was the same as previously reported (Livak and Schmittgen, 2001). The thermocycler program for RT-qPCR was 94 °C for 5 min; 40 cycles at 94 °C for 5 s, 62 °C for 15 s, 72 °C 10 s; 72 °C for 7 min. The thermocycler program for melting curves was 94 °C for 2 min with the temperature being gradually decreased to 25 °C at 0.21 °C/s, and fluorescence acquisition was continuous.

All S. cerevisiae strains were derived from the S288C genetic background. The homozygous diploid deletion mutant library were purchased from Invitrogen Inc. [http://clones.invitrogen.com/] and was frozen at − 80 °C in 96-well microtitre plates in 15% glycerol in liquid YPD medium (1% yeast extract, 2% peptone, 2% glucose). SD-URA media (0.17% (w/v) yeast nitrogen base, 2% (w/v) glucose, 0.5% ammonia sulfate, adding 1/10 mL amino acid mixture without uracil) was used to culture yeast cells for plasmid selection. Solid media were produced by adding 2% (w/v) agar when necessary. SDS was purchased from Sangon Biotech (Shanghai, China), and Dihydroethidium was purchased from Sigma (Beijing, China).

Cell lines were cultured in DMEM (HEK 293 T) or DMEM F12 (SW872, HTC75) supplemented with 10% fetal bovine serum (FBS), and 100 U/mL Penicillin/ Streptomycin (P/S). All cells were grown at 37 °C in a humidified incubator at 5% CO₂ and regularly tested for the absence of mycoplasma. Chemical reagents, such as DMSO, NaCl, SDS, and Tris base for buffer preparation were obtained from Sangon Biotech (Shanghai). The FBS, cell culture media were purchased from Hyclone. Antibodies against cyclin A2 and MCM3 were purchased form Santa Cruz Biotechnology. Antibodies against his-tag, Ki67, tubulin and flag-tag were purchased from Abcam. Antibodies against Skp2, myc-tag, p27^Kip1, rabbit IgG, and mouse IgG in western blot experiments were obtained from Cell Signaling Technology (Beverly, MA). Antibodies against Skp2, myc-tag, used in Co-IP experiment were purchased from Proteintech (Chicago, IL, USA). Anti-BrdU antibody for immunofluorescence staining was obtained from Sigma. HRP-conjugated secondary antibodies against rabbit IgG and mouse IgG were purchased from Abcam.

The expression vector pET28a(+) and E. coli strains BL21 (DE3), were stored at − 80 °C in our lab. Kanamycin, protease inhibitors phenylmethanesulfonyl fluoride (PMSF) and IPTG were purchased from Sangon Biotech (Shanghai, China). p-NPG was purchased from Bio Teke Corporation (Beijing, China). Glycine, Tris, SDS, Bromophenol blue, coomassie brilliant blue R 250 and p-nitrophenol (p-NP) were purchased from San gon Biotech (Shanghai, China). Acrylamide and TEMED were bought from Sigma (USA). Glycerinum, isopropanol, methyl alcohol, β-mercaptoethanol, ammonium persulfate and glacial acetic acid were obtained from Sinopharm Chemical Reagent (Shanghai, China). Thermo Scientific Pierce BCA Protein Assay Kit was purchased from Thermo Fisher Scientific (MA, USA). High Affinity Ni-NTA Resin was bought from Genscript (Nanjing, China). All other reagents were of analytical grade and used without any further treatment.