title: GSM8759593: B73-apex2-rep2; Zea mays; ATAC-seq
library_construction_protocol: Flash frozen and ground shoot apex 2 tissue (~0.25 g) was resuspended in 4 mL of 1x nuclei isolation buffer (16 mM HEPES; pH8, 200 mM sucrose, 0.8 mM MgCl2, 4 mM KCl, 32% Glycerol, 0.25% Triton X100, 1x complete protease inhibitor, 0.1% 2-ME, 0.1 mM PMSF), shaken at 4°C for 20 minutes and then filtered through 2 sheets of miracloth. Nuclei were centrifuged at 1,000 x g at 4°C for 15 minutes. Nuclei were resuspended in 800 µL, 400 µL, and finally 100 µL 1x TB (tagmentation buffer; 10 mM Tris Base, 5 mM MgCl2, 10% v/v dimethylformamide) and centrifuged for 5 min at 1000 x g at 4°C between each resuspension. Libraries were generated using reagents from the Illumina DNA Library Prep Kit (FC-121-1031). 2.5 µL Tn5 enzyme, 2.5 µL of 2x TB and 20 µL of nuclei were incubated for 1 hr at 37°C. 22 µL of H2O, 2.5 µL 10% SDS, and 0.5 µL of Proteinase K was added to the reaction and incubated at 55 °C for 1 hour. Tagmented libraries were purified using the Zymo clean and concentrator kit, eluting with RSB. 25 ng of tagmented DNA was combined with 2.5 µL of both index primers (Illumina; FC-121-1011), 7.5 µL PCR master mix, 2.5 µL NPM and filled to 25 µL with RSB buffer. ATAC-seq libraries were PCR amplified for 11 cycles, diluted to 50 µL and cleaned up with a two-sided Ampure XP bead size selection. A 0.5:1 bead:sample ratio followed by a 1.2:1 bead:sample ratio was used to select ~200-1000 bp libraries. ATAC-seq libraries were processed using an Illumina NovaSeq 6000 platform with a 150 bp paired-end sequencing design.