GSM3466663: ATAC-Seq ClkKO ZT04 rep1 rep2; Danaus plexippus; ATAC-seq
Brains free of eye photoreceptors were dissected in ice-cold ringer's solution and resuspended twice in 600 μl of NP-40 lysis buffer (10mM Tris-HCl at pH 7.5, 10mM NaCl, 3 mM MgCl2, and 0.1% NP-40). Crude nuclei were prepared by homogenizing the brains in a 2 ml, ice-cold, Dounce homogenizer with two strokes of a loose-fitting pestle. Nuclei was directly subjected to transposition by Tn5 transposase for 30 min at 37°C using the Nextera DNA Library Preparation Kit (Illumina), and the tagmented DNA was then purified using a Zymo DNA Clean & ConcentratorTM-5 Kit, all according to a previously published protocol (Buenrostro et al, 2015). To generate a control naked DNA library, 1ng of genomic DNA extracted with phenol/chloroform was also subjected to transposition by Tn5 transposase and the tagmented DNA was purified following the same procedures. Barcoded libraries were PCR amplified, each using a common custom primer and a unique custom Nextera barcoded primer, as in (Buenrostro et al, 2015). For each library, after an initial round of five PCR cycles, the optimal number of PCR cycles to stop amplification prior to saturation was estimated by real-time quantitative PCR. Five to ten additional PCR cycles were then performed bringing the total number to 10 (for control DNA) to 15 cycles, and the libraries were purified using a Qiagen MinElute PCR purification kit. Libraries quality and size distribution was assessed on a Bioanalyzer, libraries were quantified by real-time quantitative PCR and mixed in equimolar ratios before sequencing on a single lane of using 50bp single end reads on an Hi-seq 2500 (Illumina).
GEO Accession
GSM3466663