GSM6303847: OPA1_1; Mus musculus; ATAC-seq
Libraries were prepared using the Nextera DNA library Prep Kit (Illumina) adapting a published protocol. In vitro polarized Th17 cells were washed in PBS and then lysed for 3 min in 10 mM Tris-HCl, pH 7.4,10 mM NaCl, 3 mM MgCl2 and 0.1% Igepal CA-630, 0.1% Tween-20, 0.01% Digitonin, 1% BSA (all SIGMA). Cold wash buffer (same as lysis buffer without Igepal and digitonin) was added to the lysate and mixed three times by inverting the tubes and immediately spun 500 x g for 5 min, 4°C. Supernatant was discarded and nuclei resuspended in 2.5 μl TDE1 (Nextera Tn5, 100 nM final), 0.5 μl 1% Digitonin (0.01% final), 0.5ul 10% Tween-20 (1% final) 16.5 μl PBS and 5 μl water and incubated for 30 min at 37°C with gentle mixing (1000 RPM). DNA was purified with the QIAGEN MinElute PCR Purification Kit (Thermo Fisher Scientific). PCR amplification was performed with the NEBNext High-Fidelity 2x PCR Master Mix (New England Labs) using custom Nextera PCR Primers containing barcodes. Adaptors were removed with AMPure XP beads according to manufacturer's protocol. Libraries were quantified with the Qubit and sequenced with a HISeq 3000 (Illumina) at the Deep-sequencing Facility at the Max-Planck-Institute for Immunobiology and Epigenetics.