title: GSM5830385: BraKO_d5_rep2 (ATAC-seq); Mus musculus; ATAC-seq
library_construction_protocol: The protocol was modified from Kaestner Lab, 2020. In brief, EBs at day 5 of differentiation were trypsinised to single-cell suspension, washed with PBS and 50.000 cells were lysed in 50 µl ATAC-seq lysis buffer. Nuclei were mixed with 50 µl of transposition reaction mix containing Tagment DNA Enzyme 1 (Nextera DNA Library Preparation Kit, Illumina). The transposition reaction was carried out at 37°C and 600rpm for 30 min, purified using the Qiagen MinElute Kit and eluted in EB-buffer (Qiagen). DNA was amplified by PCR with an appropriate number of PCR cycles (5-8) using Custom Nextera PCR primer and the NEBNext High-Fidelity 2x PCR Master Mix (Nextera DNA Library Preparation Kit (New England BioLabs, E7530L) and purified with the Qiagen MinElute Kit (Qiagen, 28004). The libraries were size-selected using SPRI select beads (Beckman Coulter) to obtain fragments between <1000 bp of size. DNA concentration was measured with a Qubit Fluorometer (Thermo Fischer, Q32854) and fragment size determined with a Bioanalyzer (Agilent).