GSM8077336: Lpolyphemus_book_gills_ATAC; Limulus polyphemus; ATAC-seq
The specimen wwas euthanized by submerging in filtered seawater prepared at a salinity of 1.025 using Instant Ocean salt containing 1g/L Tricaine-S (Syndel) and buffered with 4.5 mM sodium carbonate at 25oC until the gills stopped moving. From the adult male horseshoe crab, the opisthoma was separated and book gills dissected out using shears. The exoskeleton was peeled off to reveal the cartilage tissue. The tissue was cut using scissors into 1 cm X 1 cm pieces. Each piece was flash frozen in isopentane cooled in a liquid nitrogen bath and immediately transferred into a cryovial. The flash frozen tissue was stored at -80oC for two weeks. Subsequently, 200 mg of tissue was weighed out and finely minced using ice-cold scissors. The minced tissue was transferred into ice-cold homogenization buffer (250 mM sucrose, 10 mM Tris pH 8.0, 25 mM KCl, 5 mM MgCl2, 0.1% Triton X-100, 0.5% RNAse inhibitor (Sigma; cat. no. 3335399001), 1X protease inhibitor (Roche; cat. no. 5056489001), and 0.1 mM dithiothreitol (DTT; Sigma cat. no. 646563)). Nuclei were released through mechanical digestion: a chilled dounce was used to release nuclei by 10 strokes of the loose pestle and 7 strokes of the tight pestle from the minced tissue. The dounced nuclei were strained, filtered, and washed by centrifugation at 1000 g for 10 min before fluorescence-activated nuclei sorting. We generated  barcoded single-nuclei libraries with simultaneous profiling of gene expression and chromatin accessibility using 10X Genomics Chromium Single Nuclei Multiome Reagent Kit per manufacturer's instructions.