Name: GSM6757733
Instrument: NextSeq 500
Strategy: ATAC-seq
Source: GENOMIC
Selection: other
Layout: PAIRED
Construction protocol: For ATAC-seq time course experiments, the following amounts of hand-sorted embryos were used: 400 embryos (1-1.5 h AEL); 100 embryos (1.5-2 h AEL); 40 embryos (2-2.5 h AEL, 2.5-3 h AEL). Following sorting, embryos were immediately dounced in ATAC Resuspension Buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2) with 0.1% IGEPAL CA-630 and nuclei were harvested by centrifugation. The nuclear pellet was incubated for 3 minutes on ice in ATAC resuspension buffer supplemented with 0.1% IGEPAL CA-630, 0.1% Tween-20, and 0.01% Digitonin (Promega, G9441). The reaction was stopped by adding ATAC Resuspension Buffer with 0.1% Tween-20 followed by centrifugation. Tagmentation took place at 37°C for 30 minutes at 1000 rpm in a 50 µL reaction volume containing 10 µL of 5x Tagment DNA Buffer (50 mM Tris-HCl pH 7.4, 25 mM MgCl2, 50% DMF) 16.5 µL 1x PBS, 0.5 µL 10% Tween-20, 0.5 µL 1% Digitonin, 1-2 µM assembled transposome, and water. Accessible fragments were purified using the Monarch PCR & DNA Cleanup Kit (NEB). Libraries were constructed using Illumina Nextera Dual Indexing, and qPCR was used to prevent over-amplification as done in Corces et al., 2017. ATAC-seq for chromatin accessibility using standard Illumina protocols.