GSM6606114: E14 TKO cells, D4, biological replicate 2 (ATAC_EpiLC); Mus musculus; ATAC-seq
The ATAC protocol was optimized from Fursova et al., 2021. First, 1x10^7 ESCs were mixed with 2.5x10^6 Drosophila S2 cells, and resuspended and incubated in 1ml Lysis Buffer (50mM KCl, 10mM MgSO4.7H20, 5mM HEPES, 0.05% NP40) for 5 min at RT. After a spin at 4°C 1500g for 5 min, the supernatant was discarded and nuclei pellet was resuspended in ice-cold RS Buffer (10mM NaCl, 10mM Tris-HCl, 3mM MgCl2). Nuclei were counted using Beckman Coulter ViCell XR cyto-counter, to ensure nuclei integrity. Tagmentation was performed on 50 000 nuclei per sample, with TDE1 Tn5 transposase (Nextera), at 37°C for 30 min. The reaction was stopped by the addition of PB Buffer and tagmented DNA was eluted using minElute columns from Reaction clean-up Qiagen Kit. We used 50ng of tagmented genomic DNA isolated from the same nuclei preparation to control for Tn5 transposition bias. Library preparation was made according to the manufacturer's instructions (Accel NGS 2S DNA library kit, Swift Biosciences). For library amplification, 12 PCR cycles were applied as followed: 98°C for 10 sec, 60°C for 15 sec and 68°C for one minute. Library size and quantification was assessed on Agilent 4200 Tapestation. C