title: Donor 3 Day 4
organism: Homo sapiens
description: Protocols: B-lymphocytes were isolated from anonymous human donors undergoing adenectomy. Adenoids were mechanically minced and single cells were fractionated by Ficoll Hypaque gradient centrifugation. Primary naïve B cells were sorted using a FACS Aria IIIu instrument (Becton Dickinson) to obtain pure populations of naïve B-lymphocytes (CD38- and IgD+). 1x10^6 sorted cells were collected and frozen in TRIZOL immediately after sorting to serve as the non-infected sample on day 0. The remaining cells were infected with wt EBV (2089_B95-8) with a multiplicity of infection of 0.1. At the indicated time points post infection (day 1, 2, 3, 4, 5, 8, and 14) cells (1x10^6) were sorted for intact, living cells which were frozen at -80°C in TRIZOL. Total RNA from sorted cells frozen in TRIZOL was prepared using chloroform extraction and ethanol precipitation. The RNA samples were further processed using the RNAeasy Kit (Qiagen) according to the manufacturer's instruction including a step with DNAse treatment. The first-strand cDNA was synthesized using oligo(dT) primer. PolyA enriched cDNA samples were fragmented and further used for the ligation of Illumina TruSeq sequencing adapters to the 5´ and 3 ´ends of the cDNA fragments. Barcoded cDNA samples were finally amplified by PCR reaction. Prior to sequencing the PCR amplified cDNA samples were size fractionated to obtain a range of 250-600 nt long fragments using the AMPure XP kit clean-up system from Agencourt.