GSM7885024
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). Total RNA samples of arsenate and arsenobetaine were sent to Novogene Europe, Cambridge, UK, for cDNA library preparation and sequencing using the Illumina NovaSeq 6000 platform for 150 bp paired end reads. Enriched mRNA was prepared using Oligo(dT beads). cDNA libraries were prepared following manufacturer's instructions per the Novogen pipeline (Novogene Europe, Cambridge, UK)