title: scATAC-seq of Gasterosteus aculeatus: adult gill
design: Gills were cut with sterile scissors. Gills were thoroughly washed with chilled 1  PBS and transferred to a Petri dish. The tissue was cut into small pieces with sterile scissors and washed twice with chilled 1  PBS. The pellet was trypsinized with 200 l TrypLE  Express Enzyme (Gibco, USA) for 2 minutes. One ml of fetal bovine serum (FBS; Hyclone, USA) was added to the cell suspension to inhibit trypsin activity. The cells were harvested by filtering the cell suspension through a filter (80 microns). The filtrate was centrifuged at 2000 rpm for 5 minutes. Nuclei were isolated according to the 10x Genomics protocol for Nuclei Isolation for Single Cell Multiome ATAC + Gene Expression Sequencing'' available at https://www.10xgenomics.com/. The cells were washed 2 times with 1  PBS + 0.04% BSA, and the number of cells was determined. Nuclei were isolated from 100,000-1,000,000 cells. Added 100,000-1,000,000 cells to a 2 ml microcentrifuge tube. It was centrifuged at 300 rpm for 5 min at 4  C. All supernatant was removed without destroying the cell sediment. Then we added 100 l of chilled lysis buffer (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% Nonidet P40 Substitute (if using Sigma (74385) 100% solution, prepare a 10% stock), 0.01% Digitonin (incubate at 65C to dissolve precipitate before use), 1% BSA, 1 mM DTT, 1 U/l RNase inhibitor, Nuclease-free water), and incubated for 3-5 minutes on ice. We next evaluated the efficiency of lysis using an automatic cell counter and added 1 ml of chilled wash buffer (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 1% BSA, 0.1% Tween-20, 1 mM DTT, 1 U/l RNase inhibitor, Nuclease-free water) to the lysed cells. It was centrifuged at 500 rpm for 5 min at 4  C. The supernatant was removed without disturbing the pellet of the nuclei. Based on cell concentration and assuming ~ 50% of nuclei lost during cell lysis, we resuspended in a chilled diluted nuclei buffer (1XNuclei Buffer (20X), 1mM DTT, 1 U/l RNase inhibitor, Nuclease-free water). All work procedures were performed on ice. We determined the concentration of nuclei using an automatic cell counter, and then immediately switched to Chromium Single Cell ATAC Reagent. Single-nuclei experiments were performed using a 10x Chromium Single Cell ATAC Library & Gel Bead Kit by precisely following the manufacturers detailed protocol to construct a 10x Single Cell ATAC Library. Single-nuclei libraries were run using paired-end sequencing on the HiSeq1500 platform (Illumina) according to the Chromium Single Cell ATAC Reagent Kits User Guide.