E-MTAB-6388
Transcriptome-wide mapping of signaling pathways and early immune responses in lumpfish leukocytes upon in vitro bacterial exposure
This experiment aims at mapping the early response of lumpfish leukocytes against Vibrio anguillarum exposure.   Fifteen fish were sacrificed and their head kidney were excised. From the tissue samples, leukocytes where isolated. 5x10e6 cells, from each leukocyte sample where added to each experimental sample. As it was 4 experimental groups, a total of 2 * 10e7 cells were used per fish.   The experimental groups was control 6 hours, treatment 6 hours, control 24 hours and treatment 24 hours. The control groups were added phosphate buffered saline, while the treatment groups where added 5*10 to the 7th Vibrio anguillarum O1 cells suspended in an equal amount of phosphate buffered saline.  After experiment was ended, the cells were lyzed and RNA was extracted, DNase treated, normalized and pooled . The normalization and pooling was conducted by adding 1000 nanograms RNA from 5 experimental samples to one sequencing sample.   The sequencing libraries were prepared  and sequenced by the Norwegian High Throughput Sequencing Center. . Reads of low quality, low complexity, containing adapter sequence, matching ribosomal or mitochondrial sequence, or the genomes of Vibrio anguillarum used in the stimulation experiment were discarded. Transcripts were assembled using Trinity v2.0.6. Transcript abundances were estimated using RSEM as part of the Trinity pipeline. The read count estimates were used as a basis for differential expression analysis using the Limma R-package. Only genes with at least 10 reads in at least three samples were considered for differential expression analysis.
This experiment aims at mapping the early response of lumpfish leukocytes against Vibrio anguillarum exposure.   Fifteen fish were sacrificed and their head kidney were excised. From the tissue samples, leukocytes where isolated. 5x10e6 cells, from each leukocyte sample where added to each experimental sample. As it was 4 experimental groups, a total of 2 * 10e7 cells were used per fish.   The experimental groups was control 6 hours, treatment 6 hours, control 24 hours and treatment 24 hours. The control groups were added phosphate buffered saline, while the treatment groups where added 5*10 to the 7th Vibrio anguillarum O1 cells suspended in an equal amount of phosphate buffered saline.  After experiment was ended, the cells were lyzed and RNA was extracted, DNase treated, normalized and pooled . The normalization and pooling was conducted by adding 1000 nanograms RNA from 5 experimental samples to one sequencing sample.   The sequencing libraries were prepared  and sequenced by the Norwegian High Throughput Sequencing Center. . Reads of low quality, low complexity, containing adapter sequence, matching ribosomal or mitochondrial sequence, or the genomes of Vibrio anguillarum used in the stimulation experiment were discarded. Transcripts were assembled using Trinity v2.0.6. Transcript abundances were estimated using RSEM as part of the Trinity pipeline. The read count estimates were used as a basis for differential expression analysis using the Limma R-package. Only genes with at least 10 reads in at least three samples were considered for differential expression analysis.