The CLIP-seq analyses of the motifs in the EV71 genome interacted with HSPA8 protein.

Crosslinking-immunoprecipitation and high-throughput sequencing (CLIP-Seq)At least 2 × 107 cells are needed for each repeated experiment. Vero cells were infected with mock or EV71(MOI=1) for 12 h. Then the cells were washed with cold phosphate-buffered saline (PBS) and irradiated twice at UV254 (600mJ/cm2) in a Stratalinker crosslinker (Stratagene) on ice. Cells were collected with CLIP lysis buffer containing 0.5mM DTT and EDTA-free protease inhibitor cocktail and incubated on ice for 20min then centrifuged at 14,000 × g for 15min at 4 °C and transferred supernatant into a new centrifuge tube. For immunoprecipitation procedure, the supernatant was incubated with HSPA8 antibody or IgG antibody overnight at 4 °C. The next day, the system was further incubated with Protein A/G magnetic beads for 4h at 4 °C. Afterwards, the beads were washed twice with IP wash buffer (50mM HEPES-KOH, 300mM KCl, 0.05% (v/v) NP40, 0.5mMDTT, complete EDTA-free protease inhibitor cocktail (Roche), pH 7.5) and then incubated at 37 °C for 30min and at ice for 5min. To elute protein-RNA adducts, beads were resuspended in NuPAGE SDS-PAGE loading buffer and incubated at 72 °C for 10 min, and then separated on Novex NuPAGE 10% Bis-Tris gels (Invitrogen) and transferred to nitrocellulose membranes (Amersham). For extraction of RNA-protein complexes, themembrane was treated with proteinase K buffer (100mM Tris pH7.5, 10mMEDTA, 50mMNaCl, 1% SDS, 4mg/ml proteinase K) at 55 °C for 30min with constant agitation. An equal volume of phenol: chloroform: isoamylalcohol (125:24:1) to RNA was added and precipitated with 1 μl glycogen at −20 °C for 6 h and then centrifuged at 19,000 × g for 30min at 4 °C then discarded the supernatant. The precipitate was washed twice with 75% ethanol and resuspended with 10 μl RNase-free water. The recovered RNA was used to perform high-throughput sequencing using Illumina P5 adapter and cDNA libraries were then sequenced by using DNBSEQ-T7 sequencer (MGI Tech Co., Ltd. China) under the help of Seqhealth Technology Co., LTD (Wuhan, China). This study adopted traditional methods like Multiple EM for Motif Elicitation (MEME) to discover the sequence consensus of HSPA8 binding sites in EV71 genomic RNA.<br>