Illumina HiSeq 4000 paired end sequencing; Fresh versus slow-frozen ATACseq samples for salmon tissues
Fresh versus slow-frozen ATACseq samples for salmon tissues
Four fish (Atlantic salmon, approx. 7 months old, 26-28 g) were killed with a sharp blow  to the head. Liver and brain were removed, washed with PBS and perfused to remove as much blood as possible. Small pieces of tissue was either placed directly into cold lysis  buffer (fresh tissue) or frozen in cryotubes placed on dry ice (slow frozen) Fresh and slow frozen tissues (50 mg) followed the omni-ATAC protocol for frozen tissues (1). The nuclei were counted on an automated cell counter (TC20 BioRad, range 4-6 um) and  assessed under microscope. 50 000 nuclei followed transposition for 30 minutes at 37 °C, in a shaker at 200 rpm. The samples were subsequently purified with  the MinElute PCR purification kit (Qiagen) and eluted in 12 μl of Elution Buffer.  (1) Corces MR, Trevino AE, Hamilton EG, Greenside PG, Sinnott-Armstrong NA, Vesuna S, et al. An improved ATAC-seq protocol reduces background and enables interrogation of  frozen tissues. Nat Methods. 2017 Oct;14(10):959–962. qPCR was used to determine the appropriate number of PCR cycles for  library preparation by finding the cycle number that is 1/4th of  the maximum fluorescent intensity based on the protocol of Buenrostro et al. (2).   The total number of cycles determined for library preparation ranged from 8 – 10 cycles.  Libraries were made with Nextera DNA Library Prep Kit (Illumina) and short fragments  (primer dimers) or fragments longer than 1000 bp were removed using  AMPure XP beads (Beckman Coulter, Inc.) purification. The fragment length distributions  were determined on a 2100 Bioanalyzer using the High Sensitivity Kit (Agilent) and the library concentration was determined with Qbit HS Kit (Thermo Scientific) before  submitting the samples to The Norwegian Sequencing Centre (NSC).
Experimental Factor: organism part
spleen
Experimental Factor: specimen with known storage state
frozen