Instrument: Illumina HiSeq 4000
Strategy: ATAC-seq
Source: GENOMIC
Selection: other
Layout: PAIRED
Construction protocol: Whole embryo was collected and deyolked using Ginzburg Fish Ringer solution (55mM NaCl, 1.8mM KCl, 1.25mM NaHCO3). Following trituration, 50,000 cells were collected, washed with PBS, and pelleted at 500 rcf for 5 min at 4ºC. Nuclei extracted by adding 50ul of cold lysis buffer (10 mM Tris-HCL pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% (v/v) Igepal CA-630) and immediately centrifuging at 500rcf for 10 min at 4ºC. Extracted nuclei were subjected to the transposition reaction using the Nextera Tn5 Tranposase Kit. Nuclei were incubated at 37ºC for 30 min and DNA purified using the Omega MicroElute DNA Cleanup Kit. DNA was then PCR amplified and barcoded with the NEBNext High-Fidelity 2x PCR Master Mix using the following protocol: 1. 72ºC, 5 min 2. 98ºC, 30s 3. 98ºC, 10s 4. 63ºC, 30s 5. 72ºC, 1 min; repeat steps 3-5, 5x 6. 4ºC, forever. Subsequent RT-qPCR was performed to determine additional cycles. Libraries were size selected using Mag-Bind RxnPure Plus (Omega, M1386-00) with the first selection to eliminate small products (0.4:1 ratio of beads) and the second selection (1.4:1 ratio of beads) to select desired products. Eluted samples were submitted to the BioAnalyzer to determine library size. The KAPA Library Quantification Kit (KAPA, KK4824) was used to determine library concentration prior to sequencing on the Illumina HiSeq4000.