title: GSM9168244: right brain, Day 0, fish 40E8; Gasterosteus aculeatus; ATAC-seq
library_name: GSM9168244
library_construction_protocol: Each subject was killed by rapid cervical dissection and the brain was immediately dissected under a microscope in a Sylgaard 184-lined petri-dish. Half of the brain was placed in cold PBS and Roche cOmplete Mini Protease Inhibitor Cocktail (PIC). Tissue was then cryopreserved for future use in ATACseq libraries as follows: Within 60 min of harvesting, half-brains in 350 μl of PBS with PIC kept at 4C were homogenized with a motorized pestle. They were spun down at 500x g for 5 min at 4C, then washed with 250 μl of cold PBS and spun down again at 500x g for 5 min at 4C. The pellet was gently resuspended in 300 μl of Cryostor media (Stemcell CryoStor® CS10) and frozen slowly in a Fisher Scientific Mr. Frosty isopropyl alcohol chamber at -80C. ATAC-seq libraries were prepared in batches of 10-11 samples. Samples were first thawed at 37C for 2 minutes and mixed with a pipette. Then, 3ul (1/100) of each sample was added to 1.2 ml PBS with PIC and spun down at 500x g for 5 minutes at 4C. The supernatant was discarded and the cell pellet gently resuspended in 50 μl of cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40) and spun down at 500x g for 10 minutes at 4C. The supernatant was discarded and the cell pellet gently resuspended in 50 μl of transposition reaction mix (25 μl 2x TD Buffer and 2.5 μl Tn5 Transposes from Illumina Nextera DNA Library Prep Kit and 22.5 μl Nuclease-free H2O). The samples were then incubated in a Thermomixer at 37C for 45 min with a 500 rpm mixing speed. Immediately following transposition, samples were purified using a Zymo DNA clean-up kit and eluted in 22 μl provided elution buffer. DNA fragments were amplified in a PCR reaction containing 20 μl transposed DNA, 2.5 μl Illumina Nextera i5 adapter, 2.5 μl Nextera i7 adapter, and 25 μl KAPA HiFi HotStart ReadyMix). The PCR reaction was as follows: 1) 7C for 5 min, 2) 98C for 30 sec, 3) 9°C for 10 sec, 4) 63C for 30 sec, 5) 72C for 1 min, with steps 3-5 repeated 15x. The number of PCR steps had previously been optimized for this tissue. PCR product was cleaned up using 1.8x KAPA Pure beads and eluted in 20 μl of Illumina elution buffer. Samples were quantified using Qubit fluorometry using DNA HS Assay kits and quality confirmed via Agilent 2100 Bioanalyzer using the Agilent High-Sensitivity DNA kits. One sample was determined to be of not sufficient quality. Therefore, 31 libraries were normalized using information from the Qubit and Bioanalyzer trace for a final 95.7 μl pool at 81 nM concentration submitted to Roy J. Carver Biotechnology Center at the University of Illinois Urbana-Champaign for sequencing. This pool was quality checked (using Qubit, Fragment Analyzer, and qPCR) and run on a MiSeq nano (100 nt reads) to confirm reasonably even representation of libraries. The pool was then sequenced on one lane of a NovaSeq 6000 using a NovaSeq S4 reagent kit with 2x50nt reads.