title: ATAC-seq of Atlantic salmon (Salmo salar) liver sampled at various stages through smoltification
design: ATAC-seq of Atlantic salmon (Salmo salar) liver sampled at various stages through smoltification
library_construction_protocol: Fish were euthanized by a blow to the head and samples of liver were cut into ~5 mm cubes and placed in RNAlater. Samples were incubated for at least 30 minutes at room temperature before long-term storage at -20°C. Atlantic salmon eggs, provided by AquaGen Breeding Centre Kyrksæterøra, Norway, were sterilized at the Norwegian University of Life Sciences (NMBU) fish lab and incubated at 350 to 372 day-degrees until hatching. First feeding of fry took place 5 weeks after hatching. Fry were then randomly divided into two replicate tanks and reared on a standard commercial diet high in EPA and DHA. Sampling began at 21 weeks after first feeding (week 1), and again 10, 19, and 25 weeks after that. One week after the first sampling a portion of fish from each tank were transferred to replicate light control tanks and sampled in the same manner as the experimental fish. At the same time, the experimental fish were switched to “winter-like” lighting conditions with 8 hours of light per day for 8 weeks to trigger smoltification, before returning to “spring-like” conditions with 24 hours of light per day. Immediately after the week 19 sampling, a portion of fish from each tank were transferred to seawater conditions at the Norwegian Institute for Water Research (NIVA), Solbergstranda, Norway. UV-sterilized seawater used in this life-stage had a salinity of 3%-3.5% and was obtained from the Oslofjord. Fish were sedated before transport and allowed to acclimatize for several hours before being slowly introduced to the new water conditions. The fish that were not transferred to seawater were sampled as a freshwater control at the same time as the experimental fish. The liver tissues were washed and perfused with cold PBS to remove blood before being dissociated and strained through a cell strainer. The nuclei were isolated from cell homogenate by centrifugation and counted on an automated cell counter (TC20 BioRad, range 4-6 um). Transposition of 100k (weeks 1, 19 and 25) and 75k (week 10) nuclei was performed by Tn5 transposase from Nextera DNA Library Preparation kit. The resulting DNA fragments were purified and stored at -20°C. PCR Amplification with addition of sequencing indexes (Nextera DNA CD) were done according to Buenrostro et al. 2015, with a test PCR performed to determine the correct number of amplification cycles. The ATAC libraries were cleaned by Ampure XP beads and assessed by BioAnalyser (Agilent) using High sensitivity chips. Quantity of libraries were determined by using Qubit Fluorometer (Thermo). Mean insert size for the libraries was 190 bp.