Illumina HiSeq 1500 paired end sequencing; RNA-seq of EBVwt or EBVmutant infected human primary B cells on day 1, 2, 3, and 4 post-infection
Isolated, primary B cells were isolated from human adenoid or tonsil samples and sorted for naive, resting (CD38-ive/IgD+ive) B cells. 10,000 cells were used as the uninfected control (day 0). The rest was either infected with wild type EBV or mutant EBV with an MOI: 0.1. On day 1, 2, 3, 4 post-infection viable cells were sorted based on the forward and site scatter, counted and 10,000 directly added to RLT+ plus lysis buffer with ß-mercaptoethanol. All samples were stored immediately at -80 °C until the samples of 6 biological replicates were collected. RNA extraction and cDNA library preparation is based on the prime-seq protocol. Lysates were first treated with Proteinase K (Ambion) followed by a beads clean-up (2:1 beads to lysate ratio) using solid phase reversible immobilisation (SPRI) beads (GE Biotech). A subsequent on-bead DNAseI digest was performed to remove genomic DNA, followed by another SPRI bead clean-up and reverse transcription.   Reverse transcription (RT) was performed using Maxima H- RT enzyme (Thermo Fisher Scientific) with well specific barcoded oligo dT primers and a template switch oligo (TSO). After the RT, the barcoded first strand cDNA was pooled and cleaned up using magnetic beads. Remaining oligo dT primers were removed using exonucleaseI digest. Subsequently, cDNA was PCR amplified with Kapa Hifi DNA polymerase (Roche), quantified using picogreen dye (Thermo Fisher Scientific) and quality control using capillary gel electrophoresis (Agilent Bioanalyzer 2100). Sequencing libraries were generated with the NEB NEXT Ultra II FS kit by using a custom adapter oligo and a primer, specific for the cDNA 3'- end. Fragments between 300 bp and 500 bp were selected using double size selection with SPRIselect beads (Beckman Coulter). Final libraries were quantified and quality controlled again with the Bioanalyzer 2100.