GSM3702735: Lytic_LCL_input1; Human gammaherpesvirus 4; RIP-Seq
The total RNA extraction was performed using Trizol reagent (Invitrogen, Inc., Carlsbad, CA) and treated with DNase I (Invitrogen, Inc., Carlsbad, CA). cDNA was prepared with Superscript II reverse transcriptase kit (Invitrogen, Inc., Carlsbad, CA) according to the manufacturer's protocol. The library was prepared using the TruSeq stranded mRNA kit (Illumina, San Diego, CA) according to the manufacturer's protocol. First, the elute-frag-prime stage was done at 80°C for 2 min to allow annealing without causing fragmentation [12]. RNA was reverse transcribed into first strand cDNA using reverse transcriptase and random primers. This was followed by the second strand cDNA synthesis using DNA Polymerase I and RNase H. The cDNA fragments were used for end repair process with the addition of a single 'A' base followed by ligation of adapters. The products were then purified and enriched by PCR amplification for 12 cycles to generate the final RNA-seq library. cDNA libraries were quantified and pooled and subsequent sequencing on an Illumina HiSeq3000 platform 50 bp single read sequencing module.