GSM4910616: ATAC.dom-kd.st5.rep2; Drosophila melanogaster; ATAC-seq
ATAC-Seq protocol was performed as described previously by Buenrostro et. al, 2015 with the following modifications in sample preparation: Embryos were dechorionated in 50% bleach for 2 min, and crosslinked with 5mL of fixative. The fixative consisted of bufferA (60 mM KCl, 15 mM NaCl, 15 mM HEPES [pH 7.6], 4 mM MgCl2) supplemented with 1%PFA. Embryos were fixed for 15 min in an orbital shaker at maximum speed. The crosslinking was stopped by adding a final concentration of 225 mM glycine followed by incubation for 5 min in a rotating wheel. After washing with bufferA + 0.1% Triton X100, the embryos were hand-staged on a cooling station under a microscope with transmitted light. 25 embryos were used per replicate. Fixed embryos were then resuspendend in 1ml bufferA (60 mM KCl, 15 mM NaCl, 15 mM HEPES [pH 7.6], 4 mM MgCl2) and crushed with ultrasound (Covaris E220, 45 sec, peak power 75, duty factor 10, cycles burst 200). Nuclei were pelleted by centrifugation (3200g, 10min at 4°C). We proceeded using the protocol of Buenrostro et. al, 2015. Briefly, nuclei were resuspended in transposition reaction. Samples were then incubated at 37°C for 30 min and immediately purified using the Qiagen MinElute PCR Purification Kit, according to manufacturer's instructions. Transposed DNA was eluted in 10μl of elution buffer (buffer EB, Qiagen MinElute PCR Purification Kit) and amplified as stated in Buenrostro et al., 2015 For library amplification, all the sequences of custom primers were taken from Buenrostro et al., 2013. The number of PCR cycles for each sample was determined using qPCR as recommended by Buenrostro, et al., 2015, and the remaining 45 μl PCR reaction was further amplified. DNA from these libraries was then purified using AMPure beads (Beckman Coulter, A63881) following the manufacturer's instructions.