Proteinase k

Cells were processed in the same way as co-immunoprecipitation. Lysates were divided into two equal parts: one part was incubated with anti-HA (cat. no. H6908, Sigma) or anti-Flag (cat. no. F1804, Sigma), and the other part was incubated with normal IgG, for 2 h at 4°C on a rotator. Then all samples were incubated with protein A-agarose beads (Santa Cruz) for 1 h at 4°C on a rotator. After being washed five times with RIPA, immunoprecipitated RNA-protein complexes were digested with Proteinase K (TransGen), and RNA was extracted using a RNeasy minikit (Qiagen). The reverse transcription method was the same as above. cDNA or ddH₂O was amplified with EV71 5′-UTR specific primers (forward, 5′-GCCCCTGAATGCGGCTAATC-3′, and reverse, 5′-CATGTTTGACTGTATTGAGAG-3′) and imaged by agarose gel electrophoresis.

RIP and CLIP assays were performed as previously described.^{65 (link),66 (link)} Briefly, HepAD38 cells and Huh-7 cells transfected with prcccDNA and pCMV-Cre were lysed with RIP lysis buffer. For CLIP assays, the cells were exposed to 400 mJ/cm² 254 nm ultraviolet light before lysate to enhance the binding capacity between the protein and RNA, and the lysate was treated with RNase I (Invitrogen). The cell lysates were incubated with TIAR antibody (BD Biosciences) or mouse IgG (Proteintech) embedded ProteinA beads at 4 °C overnight. After being washed five times, the beads were resuspended using NT2 buffer with RNase-free DNase I (Roche) and proteinase K (TransGen Biotech). The coprecipitated RNAs were isolated by using phenol-chloroform extraction and ethanol precipitation, and detected by quantitative real-time PCR.

Ultrapure water was produced by the TW-D24UV system of Millitrack (Haryana, India). The yeast nitrogen base without amino acids (YNB), yeast extract peptone dextrose (YPD), chitosan, and chloramphenicol were purchased from Sangon Biotech (Shanghai, China). Chinese Baijiu (52%, v/v) was purchased from a supermarket (Dalian, China). EC was procured from Sigma-Aldrich Co. (St. Louis, MO, USA). TES buffer (300 mmol/L NaCl, 50 mmol/L Tris-HCl, 25 mmol/L EDTA, 0.2% (v/v) SDS, 2 mg/mL proteinase K, pH 8.0) was purchased from TransGen Biotech (Beijing, China). Tris-saturated phenol solution (pH 8.0), absolute ethanol, sodium acetate (pH 5.3), CaCl₂, boric acid, and chloroform-isoamyl alcohol were purchased from Sangon Biotech (Shanghai, China). Glucose, glycerol, sucrose, mannose, and sorbose were purchased from Damao Co. (Tianjin, China). 4-methyl-1-pentanol was purchased from Aladdin Biotech (Shanghai, China).

DNA extraction of each batch of samples was conducted within a month after gut dissection. A CTAB (cetyltrimethylammonium bromide)/phenol-based extraction method (24 (link)) was used in DNA extraction with minor modification. Briefly, the whole gut was resuspended in a 2-mL tube containing 728 μL of CTAB buffer and 20 μL of 20 mg/mL proteinase K (TransGen Biotech). The mixture was then ground on ice using a TGrinder OST-Y 30, at 8000 rpm for 15 s, and this was repeated three times. Sterile zirconia beads (100 μL [diameter, 0.1 mm]; BioSpec, Bartlesville, OK) and 2 μL of mercaptoethanol were then added to the tubes. Tissues were vortexed using the MOBIO Vortex Genie for 3 min and then lysed by adding 5 μL of RNase A (TransGen Biotech), followed by incubation at 56°C overnight. Lysis was performed using centrifugation at 12,000 × g for 5 min, followed by transfer to a new 1.5-mL EP tube after the supernatant was removed. The pellet was mixed with 400 μL of phenol-chloroform-isoamyl alcohol (25:24:1), and the mixture was centrifuged at 12,000 × g for 15 min. The supernatant was transferred into a new 1.5-mL tube, and 50 μL of 3 M sodium acetate and 500 μL of isopropanol were added, followed by incubation at −20°C. After centrifugation at 17,000 × g for 30 min, the pellets were washed twice with 70% ethanol. Finally, DNA pellets were dissolved in 50 μL of Tris-EDTA buffer (pH 8.0).