small RNA: EBOV infected Huh7 cells, TEX treatment
EBOV-infected cells were harvested 1 day post infection. Poly(A)+ RNA was isolated from total RNA and fragmented with ultrasound (2 pulses of 30 sec at 4C). First-strand cDNA synthesis was primed with a N6 randomized primer. Then, the Illumina TruSeq sequencing adapters were ligated to the 5' and 3' ends of the cDNA. The cDNA was finally amplified with PCR using a proof reading enzyme. For Illumina sequencing, the cDNA in the size range of 400 - 600 bp was eluted from a preparative agrose gel. An aliquot of the size fractionated pools was analyzed by capillary electrophoresis. The rRNA depleted small RNA sample was poly(A)-tailed using poly(A) polymerase. The sample was then treated with Terminator exonuclease (TEX, Epicentre). Then, CAP and 5'PPP structures were removed using tobacco acid pyrophosphatase (TAP). Afterwards, an RNA adapter was ligated to the 5'-monophosphate of the RNA. First-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and the M-MLV reverse transcriptase. The resulting cDNAs were PCR-amplified using a high fidelity DNA polymerase. The cDNAs were purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and were analyzed by capillary electrophoresis.
SUB9183141
ebov_inf_small_T1439_S2