title: GSM5969801: ATACseq in EB+d9Dox(iAscl1.Lmx1b.Foxa2.V5.TRE.Pet1.HA) rep1; Mus musculus; ATAC-seq
library_construction_protocol: 50,000 cells were harvested and washed twice in cold 1X PBS. Cells were resuspended in 10 mM Tris (pH 7.4), 10 mM NaCl, 3 mM MgCl2, and 0.1% NP-40, and centrifuged immediately at 4 °C for 10 minutes. Day 9 attached neuron samples were lyzed in 0.01% NP-40 instead. The pellet was resuspended in 25 µl of 2× TD buffer, 2.5 µl TDE1 (Nextera DNA sample preparation kit, FC-121– 1030) followed by incubation for 30 min at 37 °C. The reaction was cleaned up with Min-elute PCR purification kit (Qiagen, 28004). The optimal number of PCR cycles were determined to be the one-third of the maximum fluorescence measured by quantitative PCR reaction with 1× SYBR Green (Invitrogen), custom-designed primers [45].) and 2× NEB MasterMix (New England Labs, M0541). The library was cleaned up with Min-elute PCR kit and quantified using Qubit (Life Technologies, Q32854). Nextera DNA sample preparation kit, FC-121-1030 was used for ATAC-seq. The fragment length distribution of the library was determined by Agilent High Sensitivity DNA D1000 Screentape (5067- 5585) system and the final quantification of the library before pooling was done with KAPA library amplification kit (Roche Lightcycler 480).