RNA-seq and Western blot raw data of GP-transfected and trVLP-EBOV-infected HEK293T cells
Description
Raw Western blot data and transcriptomic profiles of GP-transfected and trVLP-EBOV-infected HEK293T cells.
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Steps to reproduce
Western blotting Cells were harvested and lysed in ice-cold NP40 lysis buffer (Beyotime, P0013F) supplemented with 1× protease inhibitor cocktail (Beyotime, P1005) on ice for 30 min, with occasional vortexing. The lysates were then centrifuged at 4°C, 12,000 × g and 4°C for 10 min. The supernatant was collected, mixed with 5× SDS-PAGE loading buffer, and boiled for 10 min. Equal amounts of protein samples were separated via SDS-PAGE (120 V, 90 min), and proteins in the gel were transferred onto a nitrocellulose membrane (Merck Millipore, HATF00010) by wet electrophoretic transfer at 100 V for 90 min. The membrane was blocked in 5% non-fat milk in PBS for 1 h at room temperature, then incubated with primary antibody at 4°C overnight. After washing three times (10 min each) with PBST (Solarbio Science & Technology Corporation, T1081), the membrane was incubated with HRP-conjugated secondary antibody for 1 h at room temperature. After three additional washes with PBST, the levels of proteins were detected by an Odyssey infrared imaging system (LI-COR) or a chemiluminescence imaging analysis system (Tanon). RNA extraction, library construction, and sequencing RNA was extracted from tissue samples, and its quality was assessed by measuring concentration and purity (NanoDrop 2000; OD260/280 ≥ 1.8, OD260/230 ≥ 1.0), integrity (agarose gel electrophoresis), and RNA integrity number (RIN ≥ 7.0; Agilent 2100). Samples with RNA concentration ≥ 35 ng/μL and total RNA ≥ 1 μg were used for library preparation. Poly(A)+ mRNA was enriched using Oligo(dT)-conjugated magnetic beads and subsequently fragmented into approximately 300 bp fragments. First-and second-strand cDNA were synthesized using random hexamer primers and reverse transcriptase and DNA polymerase I, respectively. The resulting double-stranded cDNA was subjected to end repair, 3′-adenylation, and ligation to Y-shaped adapters. The libraries were amplified by 15 cycles of PCR, size-selected by 2% agarose gel electrophoresis, and quantified using TBS380 (PicoGreen). Finally, libraries were pooled and subjected to bridge amplification on a cBot system to generate clusters, followed by 2 × 150 bp paired-end sequencing on an Illumina NovaSeq 6000 platform.
Institutions
- Sun Yat-sen UniversityGuangdong, Guangzhou