GSM5903167: H1_Ngn2_0h_rep3; Homo sapiens; ATAC-seq
75,000 DIV35 neurons were washed once with 50μl cold PBS and resuspended in 50μl cold Lysis Buffer (48.5μl Resuspension Buffer (10mM Tris-HCl pH7.5, 10mM NaCl, 3mM MgCl2 in nuclease-free water), 0.1% NP-40 (Sigma/Roche 11332473001), 0.1% Tween-20, 0.01% Digitonin (EMD Millipore 300410)). The resuspension was incubated for 3 mins on ice and then 1ml of Wash Buffer (990μl Resuspension Buffer, 0.1% Tween-20) was added. Samples were centrifuged at 500g for 10 mins at 4ºC to collect the nuclei. The nuclei were then resuspended in 50μl Transposition Reaction mix (25 μl 2xTD Buffer (from Illumina Tagment DNA Enzyme and Buffer kit (Illumina 20034211)), 16.5μl 1xPBS, 0.1% Tween-20, 0.01% Digitonin, 2.5μl Tagment DNA Enzyme 1 (from Illumina Tagment DNA Enzyme and Buffer kit), up to 50μl nuclease-free water) and incubated at 37ºC for 30 mins at 1000rpm. DNA was then isolated using Qiagen MinElute Reaction Cleanup kit (Qiagen 28206) following manufacturer's instructions. The eluted ATAC DNA was amplified by PCR as previously described. Briefly, the samples were initially amplified for 5 cycles and then qPCR was performed for each sample. The relative fluorescence (Rn) was plotted against the cycle number and the additional number of cycles to perform was determined by the cycle in which the Rn reached one-third of the maximum fluorescence intensity. After PCR amplification, libraries were purified using Qiagen MinElute PCR Purification kit and eluted in 20 μl of Elution Buffer. Libraries were sent to Genewiz for sequencing using HiSeq.