NDV-12h
Homo sapiens
Protocols: NDV-infected HeLa cells were trypsinized with 0.5 mL of trypsin (2μg/mL) (Sigma, USA) at 6 and 12 h post-infection (hpi) respectively and centrifuged at 1200g for 8 min and washed three times with ice-cold PBS. Sham infected cells were used as the mock group and were collected as the same way as NDV-infected cells. HeLa cells (ATCC CCL-2) were cultured in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, USA) supplemented with 10% fetal bovine serum (FBS) (Life Technologies, USA), 100 U/mL penicillin (Invitrogen, USA) and 100 μg/mL streptomycin (Invitrogen, USA) at 37◦C under 5% CO2 atmosphere. HeLa cells were infected with NDV strain Herts/33 at a multiplicity of infection (MOI) of 1 for 1h in serum-free DMEM. Then, the cells were washed three times with phosphate-buffered saline (PBS) and incubated at 37℃ in DMEM supplemented with 2% FBS. Three replicates of each group were prepared and pooled separately for total RNA extraction using the EasyPure RNA kit (TransGen Biotech, China) according to the manufacturer's instructions. The total concentration and purity of total RNA samples were measured with a Nano Drop ND-2000 spectrophotometer (Thermo Fisher Scientific, USA) and an Agilent 2200 Bioanalyzer (Agilent Technologies, USA). The total RNA from each sample was sequentially ligated to 3' and 5' small RNA adapters. Then, cDNA was synthesized and amplified using TruSeq Small RNA Sample Preparation Kit (Illumina, USA) according to the manufacturer's instructions. 145-160 bp PCR-amplified fragments were extracted and purified from the PAGE gel.