GSM6482255: Toll10B 3h ATAC Replicate 2; Drosophila melanogaster; ATAC-seq
Hand-sorted embryos were dechorionated in dilute bleach, rinsed thoroughly in embryo wash buffer (PBS, 0.1% Triton X-100) and crude nuclear extracts isolated by homogenizing the embryos using a motor pestle in ATAC lysis buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% IGEPAL CA-630) and centrifugating at 700 g for 10 min. The nuclear pellet was resuspended in 22.5 μl of ATAC lysis buffer, 2.5 μl Tn5 (Tagment DNA Enzyme 1 (TDE) (Illumina)) and 25 μl Tagment DNA Buffer (Illumina) and subjected to tagmentation at 37°C on a thermomixer at 1,000 rpm. Transposition was blocked by the addition of 1% SDS and DNA purified with Agencourt AMPure XP beads (Beckman Coulter, A63881) according to the manufacturer's instructions using a 2:1 ratio of beads to sample. Libraries were prepared as previously described in 'Buenrostro, J.D., Giresi, P.G., Zaba, L.C., Chang, H.Y., and Greenleaf, W.J. (2013). Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome positions. Nat Methods 10, 1213-1218.'. Briefly, tagmented DNA was PCR amplified using 1x Phusion® High-Fidelity PCR Master Mix with GC Buffer (NEB) and 1.25 μM i5 and i7 PCR primers (Nextera® Index Kit (Illumina)) with the following PCR amplification conditions: 72°C for 5 min, followed by 10 cycles of 98°C for 10 seconds, 65°C for 1 min and 15 seconds, then 72°C for 1 min. Amplified libraries were purified with Agencourt AMPure XP beads with a 1.5:1 ratio of bead to sample volume. Libraries prepared from biological triplicates were sequenced paired-end (2 x 150 bp) on the Illumina NovaSeq platform at SciLifeLab, Stockholm. ATAC-seq reads from each sequenced library were mapped to the Drosophila melanogaster (dm6) genome assembly using Bowtie2 with the default parameters. RPKM normalized ATAC-seq coverage tracks were generated using the deepTools (v.3.5.1) package. Bigwig files of the mean RPKM were produced using the deepTools 'bigwigCompare' tool.