Instrument: Illumina NovaSeq 6000
Strategy: ATAC-seq
Source: GENOMIC
Selection: other
Layout: SINGLE
Construction protocol: Briefly, ~10 eye-antennal discs were dissected and lysed using 50 µl of cold ATAC-Resupension Buffer (RSB) (see Corces et al. for composition) containing 0.1% NP40, 0.1% Tween-20 and 0.01% digitonin by pipetting up and down three times and incubating the cells for 3 min on ice. The lysis was washed out by adding 1 mL of cold ATAC-RSB containing 0.1% Tween-20 and inverting the tube three times. Nuclei were pelleted at 500 RCF for 10 min at 4°C, the supernatant was carefully removed and nuclei were resuspended in 50 µl of transposition mixture (25 µl 2x TD buffer (see Corces et al. for composition), 2.5 µl transposase (100 nM), 16.5 µl DPBS, 0.5 µl 1% digitonin, 0.5 µl 10% Tween-20, 5 µl H2O) by pipetting six times up and down, followed by 30 minutes incubation at 37°C at 1000 RPM mixing rate. After MinElute clean-up and elution in 21 µl elution buffer, the transposed fragments were pre-amplified with Nextera primers by mixing 20 µl of transposed sample, 2.5 µl of both forward and reverse primers (25 µM) and 25 µl of 2x NEBNext Master Mix (program: 72°C for 5 min, 98°C for 30 sec and 5 cycles of [98°C for 10 sec, 63 °C for 30 sec, 72°C for 1 min] and hold at 4°C). To determine the required number of additional PCR cycles, a qPCR was performed (see Buenrostro et al.83 for the determination of the number of cycles to be added). The final amplification was done with the additional number of cycles, samples were cleaned-up by MinElute and libraries were prepped using the KAPA Library Quantification Kit as previously described82. Samples were sequenced on a NextSeq500 High Output chip, with 50bp single-end reads. Omni-ATAC-seq (Corces et al., 2017)