E-MTAB-3632:RP2
Illumina HiSeq 2500 paired end sequencing; Common frog (Rana temporaria) transcriptomes following Exposure to Ranavirus and Batrachochytrium dendrobatidis
Common frog (Rana temporaria) transcriptomes following Exposure to Ranavirus and Batrachochytrium dendrobatidis
RP2
R. temporaria eggs were obtained from a private garden pond in Chessington in the UK with the permission of the landowner. This site has a known history of Ranavirus infection, but an unknown history of Bd infection. The eggs were hatched and reared through metamorphosis under controlled conditions, showing good survival and no signs of infection. Metamorphs (n = 45) were moved into an experimental room (18-21ºC, 33-46% humidity, full spectrum UV light) for acclimatisation one week prior to exposure, and were placed in individual boxes and fed on crickets ad libitum. Livers were used because they are large, easily targeted organs which are important for immunity and are a target organ in ranavirus disease. Livers were immediately dissected, preserved individually in RNAlater solution (Sigma Aldrich), and stored at -80ºC. Ranavirus (RUK13 isolate) was cultured at 24ºC in Fathead Minnow cells (FHM, Epithelial-like cells from the posterior anal tissue, obtained from the European Collection of Cell Cultures catalogue number 88102401) in maintenance media (EMEM + 10% FBS + 1% L-Glutamine) and quantified on 96 well flat-bottomed cell culture plates using the TCID50 method. Bd inoculum (Isolate IA 042, BdGPL) was prepared by culture in mTGhL medium at 18ºC for four days before counting zoospores using a haemocytometer.   This work was carried out under Home Office license (Project Licence numbers PPL 80/2214 and PPL 80/2466) and was approved by the Institute of Zoology Ethics Committee and the University of Exeter Ethical Review Board. In total 45 frogs were used - 15 animals for each of three exposure treatments (Ranavirus, Bd, Control). Animals were checked, cleaned and given fresh water frequently whilst being mindful of causing them distress through unnecessary handling. Exposure was performed in individual tubes with 29ml aged tap water with 1ml of inoculum (Ranavirus at 1k TCID50/ml or Bd at 100k active zoospores/ml), or 30ml tap water for the control treatment, for four hours. Following exposure, the frogs were returned to their individual boxes and examined daily for signs of Ranavirus infection (oedema of the eye, skin ulceration, bleeding), which would have served as an endpoint for the experiment but none were observed. Frogs were euthanized according to Schedule 1 to the Animals (Scientific Procedures) Act 1986 four days post-exposure by immersion in fresh 5g/L Tricaine methane sulphonate (MS222, Pharmaq Ltd.) solution neutralized with sodium bicarbonate in accordance with Universities Federation for Animal Welfare guidance to ameliorate suffering.  Total RNA was extracted from 5-20µg of liver tissue using the Qiagen RNeasy Mini Kit (Qiagen, Valencia USA) using the standard protocol. Each treatment consisted of 15 frogs; three pools were sequenced per treatment, with five individuals per pool. We therefore prepared nine samples for sequencing; 3 replicates per treatment, each consisting of pooled RNA extracted from the livers of 5 individuals. RNA concentration was estimated using a NanoDrop (NanoDrop, Wilmington USA), and concentrations of individual sample extractions were equalized within pools, resulting in a total pool volume of 80µl (concentrations varied between pools). The sample concentrations were not normalized.  1µl ERCC spike in control mix (Invitrogen) was added to each sample before library preparation.  Poly-A-RNA was isolated and barcoded RNAseq libraries were prepared using ScriptSeq v2 (Epicentre) with 15 cycles PCR amplification. Equimolar amounts of two libraries were pooled denatured and diluted to 6 pM
UCL
E-MTAB-3632:RP2
Experimental Factor: Ranavirus exposure
stimulus