title: HSV1_2
design: Total RNA was isolated and purified via TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. The RNA amount and purity of each sample were quantified via a NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). The RNA integrity was assessed with a Bioanalyzer 2100 (Agilent, CA, USA) with a RIN > 7.0 and confirmed by electrophoresis with a denaturing agarose gel. Poly(A) RNA was purified from 1 mg of total RNA via Dynabeads Oligo (dT)25-61005 (Thermo Fisher, CA, USA) via two rounds of purification. Then, the poly(A) RNA was fragmented into small pieces via the Magnesium RNA Fragmentation Module (NEB, USA) at 94C for 5~7 min. The cleaved RNA fragments were subsequently reverse transcribed to create cDNA via SuperScript II Reverse Transcriptase (Invitrogen, USA), which was subsequently used to synthesize U-labeled second-strand DNAs with E. coli DNA polymerase I (NEB, USA), RNase H (NEB, USA) and dUTP Solution (Thermo Fisher, USA). An A-base is then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contains a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Single- or dual-index adapters were ligated to the fragments, and size selection was performed with AMPureXP beads. After heat-labile UDG enzyme (NEB, USA) treatment of the U-labeled second-strand DNAs, the ligated products were amplified via PCR under the following conditions: initial denaturation at 95C for 3 min; 8 cycles of denaturation at 98C for 15 sec, annealing at 60C for 15 sec, and extension at 72C for 30 sec; and a final extension at 72C for 5 min. The average insert size for the final cDNA library was 300  50 bp.
library_name: R008
identifiers.namespace: SUB14867135
identifiers.id: R008