small RNA: Mock Huh7 cells, TEX treatment
EBOV-infected cells were harvested 1 day post infection. Small RNA species < 200 nt were isolated using the RNeasy Mini Kit (Qiagen). All small RNA samples were treated with Terminator Exonuclease (TEX, Epicentre) in order to degrade RNA species which carry a 5' mono-phosphate (5'P) and enrich for primary transcripts. CAP structures at the 5' ends of the small RNAs were then converted into 5'P using CapClip Acid Pyrophosphatase (Cellscript). The TEX and CapClip treated RNA samples were separated on a denaturing 10% polyacrylamide (PAA) gel. The small RNA fractions with a length below 100 bases were obtained by a passive elution of the RNAs from the gels. The eluted RNAs were then precipitated with ethanol and dissolved in water. Oligonucleotide adapters were ligated to the 5' and 3' ends of the sRNA samples. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3 adapter as primer. The resulting cDNAs were amplified with PCR using a high fidelity DNA polymerase. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and was analyzed by capillary electrophoresis.
SUB9183141
ebov_mock_small_T1757_S2