title: GSM3499319: ATACseq_stimulated_WT_rep2; Mus musculus; Tn-Seq
library_construction_protocol: 5x104 HoxER-PU.1 WT and HoxER-PU.1 KO neutrophils were harvested after 2 hour stimulation with heat-inactivated C. albicans material (MOI=1) or treament for 2h with DMSO, 1h with TSA [100 nM] and 2h with Entinostat [5 µM]. All centrifugations steps were carried out at 500 g at 4°C in a fixed-angle centrifuge. Cells were washed once in 50 μl cold PBS and were lysed in 50 μl cold ATAC lysis buffer (10 mM NaCl, 10 mM Tris, pH 7.4, 3 mM MgCl2, 0.1 % Igepal CA 630) while centrifuging for 10 min. The supernatant was discarded, and the crude nuclei extract (pellet) was resuspended in 50 μl transposition mix composed of 2.5 μl Tn5 Transposase, 25 μl tagmentation buffer (Nextera Sample Prep Kit) and 22.5 μl H2O. The mixture was incubated for 30 min at 37°C in an Eppendorf Thermomixer at 300 rpm. The reaction was purified using the MinElute kit following instructions of Qiagen and eluted in 10 μl of prewarmed TE buffer. The transposed DNA was amplified for 12 cycles using preactivated (1 min at 98°C before use) KAPA HiFi Hotstart Ready Mix and single-index primers following the Nextera Low Plex Pooling guidelines. To obtain PCR-amplifiable fragments, PCR was initiated with an elongation step to obtain amplifiable product since the transposase inserts adapters only to the 5' end of each fragment. PCR was run as followed: holding stage (5 min @70°C; 3 min @95°C) and cycling stage (20 sec @98°C, 30 sec @ 63°C, 15 sec @ 72°C). PCR amplified samples were purified using AMPure XP beads (Beckman Coulter) at a beads/sample ratio of 1.2x. Library quality control was performed with an Agilent High-Sensitivity DNA chip on a Bioanalyzer 2100. The Core Facility Genomic Muenster performed library quantification by qPCR (Kapa Library Quant Kit) and paired-end sequencing on an Illumina Nextseq 500.