small RNA: EBOV infected Huh7 cells
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. 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_S3