title: GSM5419683: ATAC-seq_Control replicate 2; Danio rerio; ATAC-seq
library_construction_protocol: FAC sorted GFP+ cells were gently centrifuged and Hank's solution was replaced by lysis buffer (10mM tris-HCL, pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% IGEPAL CA-630). Cells were immediately centrifuged at 500 g for 10minutes at 4oC. The supernatant was discarded and replaced with the transposition reaction mix (Tn5 in TD buffer) for tagmentation, and incubated at 37oC for 30min. Afterwards, 500mM of EDTA was used for quenching. The solution was incubated for 30 minutes at 50oC. MgCl2 was added to a final concentration of 45mM. Samples were stored at 4oC before proceeding with PCR amplification PCR amplification we used 1.25µl IDT® for Illumina Nextera DNA Unique Dual Indexes Set C, which contains two indexes premixed and 25 µL of Bioline MyFi Mix. This is in the place of the NEB Next HiFi PCR mix in your protocol. We performed the PCR as outlined. For PCR amplification 15 cycles were used due to the reduced amount of material. The amplified library was purified using the Qiagen PCR purification MinElute kit. This was followed by a 1 x volume AMPure XP bead-based clean-up according to manufacturer's guidelines. The resulting libraries were evaluated for quantity and quality using a Thermo Fisher Scientific Qubit 4.0 fluorometer with the Qubit dsDNA HS Assay Kit and an Advanced Analytical Fragment Analyzer System using a Fragment Analyzer NGS Fragment Kit, respectively. The ATAC-Seq libraries were further quantified by qPCR using a Bioline JetSeq library Quantification Lo-ROX kit according to their guidelines. The libraries were pooled equimolar and further cleaned using AMPure XP beads as described above. The library pool was then again assessed for quantity and quality using fluorometry and capillary electrophoresis as described above.