GSM7870430: F3-SugControl 48h; Salmo salar; RNA-Seq
Hepatocytes for RNA extraction and sequencing were seeded on 6-well Alvetex® scaffolds. After 48 h exposure to arsenite, arsenate and arsenobetaine, 2 mL of cell medium from each well was taken and kept at -20ºC until further analysis (see section 2.6). The rest of the cell medium was discarded. Retrieval of cells from Alvetex® scaffolds for this analysis was performed according to manufacturer's description (see protocol for retrieval of cells from Alvetex® scaffold's for metabolomic analysis). In summary, the well inserts with the hepatocyte cells were transferred to new 6-well plates. The cells were washed with PBS (1 mL) which was removed from the well before the next step. The Alvetex Scaffold membrane was removed with the aid of flat-ended forceps. To break down the cells, introduce 600 µL of the Qiagen RNeasy® kit lysis buffer RLT directly to the well where is Alvetex Scaffold membrane was left. Allow the plate on a rotating platform (100 rpm) for 10 min at room temperature. Conclude the process by adding 600 µL of 70% ethanol to the now-homogenized lysate. Ensure a proper mix by pipetting the combination up and down around 10 times. The cell suspension was transferred to centrifugation tubes and centrifuged at 1000×g for 5 min at 4 °C. Cell pellets were flash frozen and stored at −80 °C until RNA extraction was performed.Total RNA was extracted from Atlantic salmon Primary hepatocytes using a RNeasy Plus Mini Kit (Qiagen, Crawley, UK) as previously described (Søderstrøm et al 2023). Total RNA purity was evaluated using a nanodrop NanoDrop™ (Thermo Fisher Scientific™, MA, USA) Samples had a 260/230 and 260/280 ratio of 2.2 ± 0.1 and 2.09 ± 0.03 respectively. Integrity of RNA for each sample was investigated using RNA 6000 Nano LabChips and an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Although the integrity of the majority of the samples were assessed as good (RIN = 9.7 ± 0.12, N = 48), RNA from cells exposed to 250 µM AsSug showed a reduction in integrity (RIN 5.3 ± 0.5, N = 6). RNAseq of arsenite and arsenosugar 328 samples was conducted by a commercial company (Azenta Life Sciences, Leipzig, Germany). RNA samples were quantified using Qubit 4.0 Fluorometer (Life Technologies, Carlsbad, CA, USA). The RNA sequencing library was prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina using manufacturer's instructions (New England Biolabs, Ipswich, MA, USA). Briefly, mRNAs were initially enriched with Oligod(T) beads. Enriched mRNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3'ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. The sequencing libraries were validated using DNA Kit on the Agilent 5600 Fragment Analyzer (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 4.0 Fluorometer (Invitrogen, Carlsbad, CA). The sequencing libraries were multiplexed and clustered onto a flow-cell on the Illumina NovaSeq 6000 instrument according to manufacturer's instructions. The samples were sequenced using a 2x150bp Paired End (PE) configuration. Image analysis and base calling were conducted by the NovaSeq Control Software (NCS). Raw sequence data (.bcl files) generated from Illumina NovaSeq was converted into fastq files and de-multiplexed using Illumina bcl2fastq 2.20 software. One mis-match was allowed for index sequence identification.