title: Sample 13
organism: Cyclopterus lumpus
description: Protocols: Farmed lumpfish (C. lumpus L.) (weight 332.4 ± 63 g, length 17.6 cm ± 0.97 g) were obtained from a commercial breeder in Sogn & Fjordane County, Norway (Fjord Forsk Sogn AS). During quarantine, the fish were screened for pathogens before being reared in a 500L tank at the Bergen High-Technology Centre's Aquatic and Industrial Laboratory (ILAB) under normal rearing conditions with a 12h light: 12h dark light regime. The temperature of the outlet water was 8 °C, the salinity was 34 PSU, and the minimum oxygen saturation was 77%. The fish were fed with Amber Neptune commercial dry feed (1.5mm) at 2% bodyweight. The head kidney was isolated from healthy lumpfish and homogenized as described in our previous study(38). Briefly, the leukocytes were separated using discontinuous Percoll gradients a described previously (38, 39). Isolated leukocytes were resuspended in L-15+ medium ((L-15 media without L-Glutamine adjusted to 370 mOsm by adding 5% (v/v) of a solution consisting of 0.41 M NaCl 0.33 M NaHCO3 and 0.66 5 (w/v) D-glucose) supplemented with 100 μg/ml genatamicin (Lonza Biowhittaker Verviers, Belgium), 10 U/ml heparin (Lonza Biowhittaker Verviers, Belgium) and 15 mM HEPES (Sigma-Aldrich, St. louis, USA). Using a CASY TT Cell Counter (Innovatis AG, Reutlingen, Germany), the number of cells, cell viability, and aggregation factor were measured. Leukocytes from each of the 15 fish as distributed into four samples (stimulated samples which were exposed to poly(I:C)for 6 and 24 hours, and non-stimulated controls for the two time points) in 24 well plates (NUNC). Poly(I:C)(100 µg mL-1) in a total volume of 0.5 mL were added to the stimulated samples, while for non-exposed cells, L-15+ medium was added instead of poly (I:C). Following incubation at 15°C for 6 and 24 hours, the plates were centrifuged for 10 minutes at 200 x g. The supernatants were removed and lysis buffer containing β-mercaptoethanol was added to each well. The lysates were stored at -80 °C before RNA isolation. On average 24383597 (SE 906038) read pairs per sample passed the quality control steps, these reads were then aligned to the Cyclopterus lumpus genome, fCycLum1.pri, from Ensemble Release 109 (ensembl.org) using STAR 2.7.10 (40) and the with an average unique alignment rate of 70.2%. Gene counts were obtained using HTSeq 2.0 (41) using the C. lumpus gene annotations from Ensembl Release 109.Differentially expressed genes (DEGs) were determined using DESeq2 (42) between exposed and non-exposed samples at both 6 and 24 hours separately as well between all exposed and non-exposed samples using DESeq2's functions to account for multi-factor model design. Leukocytes from each of the 15 fish as distributed into four samples (stimulated samples which were exposed to poly(I:C)for 6 and 24 hours, and non-stimulated controls for the two time points) in 24 well plates (NUNC). Poly(I:C)(100 µg mL-1) in a total volume of 0.5 mL were added to the stimulated samples, while for non-exposed cells, L-15+ medium was added instead of poly (I:C). Following incubation at 15°C for 6 and 24 hours, the plates were centrifuged for 10 minutes at 200 x g. The supernatants were removed and lysis buffer containing β-mercaptoethanol was added to each well. The lysates were stored at -80 °C before RNA isolation. Leukocytes from each of the 15 fish as distributed into four samples (stimulated samples which were exposed to poly(I:C)for 6 and 24 hours, and non-stimulated controls for the two time points) in 24 well plates (NUNC). Poly(I:C)(100 µg mL-1) in a total volume of 0.5 mL were added to the stimulated samples, while for non-exposed cells, L-15+ medium was added instead of poly (I:C). Following incubation at 15°C for 6 and 24 hours, the plates were centrifuged for 10 minutes at 200 x g. The supernatants were removed and lysis buffer containing β-mercaptoethanol was added to each well. The lysates were stored at -80 °C before RNA isolation. On average 24383597 (SE 906038) read pairs per sample passed the quality control steps, these reads were then aligned to the Cyclopterus lumpus genome, fCycLum1.pri, from Ensemble Release 109 (ensembl.org) using STAR 2.7.10 (40) and the with an average unique alignment rate of 70.2%. Gene counts were obtained using HTSeq 2.0 (41) using the C. lumpus gene annotations from Ensembl Release 109.Differentially expressed genes (DEGs) were determined using DESeq2 (42) between exposed and non-exposed samples at both 6 and 24 hours separately as well between all exposed and non-exposed samples using DESeq2's functions to account for multi-factor model design. GeneElute Mammalian Total RNA miniprep kit (Sigma) was used to isolate total RNA in accordance with the manufacturer's instructions. Samples were treated with DNase I (Sigma) to eliminate any lingering traces of genomic DNA. One RNA sample for the RNA sequencing was made by combining total RNA (1 µg) from five fish. Three parallels were set up for RNA sequencing for each time point. After being cleaned with RNA clean & concentrator-5 (Zymo Research) in accordance with the manufacturer's instructions, the RNA was tested for quality in an Agilent 2100 bioanalyzer. The RIN values varied from 8.5 to 10 The TruSeq Stranded mRNA sample Preparation kit (Illumina®) was used by the Norwegian High Troughput Sequencing Centre to create sequencing libraries using dual indexing on a Perkin Elmer Sciclone NGSx liquid handler system. Final libraries were checked for size and adapter contamination using a standard sensitivity Fragment analyzer NGS kit, and library concentration using qPCR with Kapa Library quantification kit for Illumina (Kapa Biosciences). Paired RNA sequencing was performed on an Illumina HiSeq4000.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2023-05-13T00:14:53Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2023-05-13T00:14:53Z
attributes.tag: External Id
attributes.value: SAMEA113399845
attributes.tag: INSDC center name
attributes.value: Postdoctoral Researcher
attributes.tag: INSDC first public
attributes.value: 2023-05-13T00:14:53Z
attributes.tag: INSDC last update
attributes.value: 2023-05-13T00:14:53Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-12884:Sample 13
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell_type
attributes.value: leukocyte
attributes.tag: common name
attributes.value: lumpfish
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: organism part
attributes.value: head kidney
attributes.tag: sample name
attributes.value: E-MTAB-12884:Sample 13
attributes.tag: scientific_name
attributes.value: Cyclopterus lumpus
attributes.tag: stimulus
attributes.value: poly(I:C) 100 microgram per milliliter