title: GSM5335561: 16-T2_S11_ATAC-Seq; Mus musculus; ATAC-seq
library_construction_protocol: DNA was extracted from purified dissected mouse embryonic MNs. Cells were aliquoted and washed twice in ice-cold 1Ã— PBS. Cell pellets were resuspended in 10 mM Tris (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40 (v/v), 0.1% tween20, 0.01% Digitonin and 1% BSA, centrifuged at 500 g for 5 min at 4°C. Pellets were resuspended in 12.5 ul of 2Ã— tagmentation DNA buffer, 1.25 ul Tn5 (Nextera DNA Sample Preparation Kit, FC-121€“1030) and 11.25 ul of water, and incubated at 37°C for 30 min. The sample was purified using the MinElute PCR Purification Kit (Qiagen, 28004). PCR enrichment of the library was performed with custom-designed primers and 2Ã— NEB Master Mix. A qPCR reaction with 1Ã— SYBR Green (Invitrogen), custom-designed primers and 2Ã— NEB Master Mix (New England Labs, M0541) was performed to determine the optimal number of PCR cycles (one third of the maximum measured fluorescence) (Buenrostro et al., 2013). The libraries were purified using the AMPure XP beads (Beckman Coulter, A63880). High Sensitivity DNA ScreenTape (Agilent, 5067-5584) was used to verify the fragment length distribution of the library. Library quantification was performed using the KAPA Library Amplification kit on a Roche LightCycler 480. The libraries were sequenced on an Illumina NovaSeq (100 cycles, paired-end)