Donor 1 Day 2
Homo sapiens
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.
ENA-FIRST-PUBLIC
2019-07-01T17:03:55Z
ENA-LAST-UPDATE
2019-03-22T12:36:21Z
External Id
SAMEA5529886
INSDC center name
Research Unit Gene Vectors Helmholtz Zentrum Munchen German Research Center for Environmental Health and German Center for Infection Research (DZIF), Partner site Munich, Germany
INSDC first public
2019-07-01T17:03:55Z
INSDC last update
2019-03-22T12:36:21Z
INSDC status
public
Submitter Id
E-MTAB-7805:Donor 1 Day 2
broker name
ArrayExpress
cell type
B-lymphoblast
common name
human
individual
Donor 1
sample name
E-MTAB-7805:Donor 1 Day 2
scientific_name
Homo sapiens