title: Ion Torrent S5 XL sequencing; The Transcriptional Landscape of Marek's Disease Virus in Primary Chicken B Cells Reveals Novel Splice Variants and Genes
design: The Transcriptional Landscape of Marek's Disease Virus in Primary Chicken B Cells Reveals Novel Splice Variants and Genes
library_construction_protocol: Embryonated SPF Valo chicken eggs (VALO BioMedia GmbH,) were used for the preparation of chicken embryo cells (CEC). CEC were maintained in minimal essential medium (MEM, PAN Biotech; Aidenbach, Germany) supplemented with 1-10% fetal bovine serum (FBS) and penicillin/streptomycin. B cells were obtained from the bursa of Fabricius by dissociation of the organ and subsequent isolation of the cells by density gradient centrifugation. Briefly, the bursa of Fabricius was homogenized through a 40 μm cell filter to obtain a uniform single cell suspension. Suspension cells were carefully applied on a Biocoll separating solution (Biochrom; Berlin, Germany), centrifuged for 12 min at 650 × g with slow acceleration, and deactivated deceleration. All viruses were reconstituted by calcium phosphate transfection of CEC with purified bacterial artificial chromosome (BAC) DNA. The very virulent RB1B strain and the vaccine strain CVI988 both express a green fluorescent protein (GFP) under the control of the early thymidine kinase promotor. The viruses were propagated on CEC for up to six passages, and infected cells were stored in liquid nitrogen until further use. All virus stocks were titrated on fresh CEC. Lymphocytes at the interphase were carefully transferred to a new tube, washed with PBS, and maintained in RPMI 1640 (PAN Biotech) supplemented with 10% FBS and penicillin [100 U/ml]/streptomycin [100 µg/ml] at 41°C under a 5% CO2 atmosphere. B cells were activated using recombinant soluble chicken CD40 ligand (chCD40L), which was expressed in HEK293 cells and purified using a Vivacell 250 ultrafiltration concentrator (Sartorius; Göttingen, Germany). Primary chicken B cells were infected by co-cultivation with infected CEC due to the strictly cell-associated nature of MDV. One million CEC were infected with 30,000 plaque-forming units (PFU) of CVI988, RB1B, or mock infected. After 4 days, one million B cells were seeded on the infected 6-well-plates in the presence of CD40L for 16 hours at 41°C. All B cells were then carefully removed from the CEC monolayer, washed with phosphate buffered saline (PBS) and prepared for fluorescence-activated cell sorting. Total RNA was extracted using TRIzol reagent (Life Technologies; Carlsbad, CA, USA) in combination with the RNeasy Mini Kit (Qiagen; Hilden, Germany) following the manufacturer's instructions. Additionally, RNA was treated with DNase using the RNase-Free DNase Set (Qiagen). Subsequently, ERCC ExFold RNA Spike-In mix 1 (Invitrogen; Carlsbad, CA, USA) was added to the total RNA as an internal control and the polyadenylated (poly(A)) RNA fraction was extracted using the Dynabeads mRNA DIRECT Micro kit (Invitrogen).  Whole transcriptome libraries were prepared using the Ion Total RNA-Seq Kit v2 (Life Technologies) following the manufacturer's instructions. Quality and quantity of the nucleic acids was controlled at each step using the NanoDrop 1000 spectrophotometer (Peqlab) or Agilent 2100 Bioanalyzer (Agilent Technologies; Böblingen, Germany) in combination with appropriate chips, respectively.
attributes.tag: Experimental Factor: infect
attributes.value: Gallid alphaherpesvirus 2 attenuated CVI988 strain