GSM5176436: Kc167_mock2_ATACseq; Drosophila melanogaster; ATAC-seq
ATAC-seq was performed following protocol from Kaestner Lab (https://www.med.upenn.edu/kaestnerlab/assets/user-content/documents/ATAC-seq%20Protocol%20(Omni)%20-%20Kaestner%20Lab.pdf) with minor modification. 100,000 Kc cells were washed with 50 μl cold 1X PBS and followed and discard supernatant. Cell pellet was lysed with 50 μl cold lysis buffer (10mM Tris-HCl, pH 7.5, 10mM NaCl, 3mM MgCl2, 0.1% NP-40, 0.1% Tween-20, 0.01% Digitonin) and incubate for 10 min in ice. Then 500 μl of wash buffer (10mM Tris-HCl, pH 7.5, 10mM NaCl, 3mM MgCl2, 0.1% Tween-20) was added to lysate. Then nuclei were collected by centrifuging at 500 g for 10 min at 4°C and resuspended nuclei in transposition reaction mix [25 μl 2X TD buffer (Illumina), 16.5 μl PBS, 0.5 μl 10% Tween-20, 0.5 μl 1% Digitonin, 2.5 ul Tn5 enzyme (Illumina), 5 ul nuclease free water) and incubated for 45 min at 37°C at 1000 rpm (Eppendorf Thermomixer) for fragmentation. DNA was purified with Qiagen Minelute columns and libraries were amplified by adding 10 ul DNA to 25 ul of NEBNext HiFi 2x PCR mix (New England Biolabs) and 2.5 ul of 25 uM each of Ad1 and Ad2 primers using 11 PCR cycles. Libraries were purified with 1.2x AMPure XP beads. All samples were sequenced with NextSeq-550 (Illumina) using 50 bp paired-end sequencing.