ESC medium with 1ug/ml doxycycline and 50ug/ml ascorbic acid
marker
sorted for SSEA+
batch
EC-DG-4663
Sequenced DNA Library
library_strategy
ATAC-seq
library_source
GENOMIC
library_selection
other
library_construction_protocol
50,000 cells were washed once with 50 μL of cold PBS and resuspended in 50 μL lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.2% (v/v) IGEPAL CA-630). The suspension of nuclei was then centrifuged for 10 min at 800 g at 4°C, followed by the addition of 50 μL transposition reaction mix (25 μL TD buffer, 2.5 μL Tn5 transposase and 22.5 μL nuclease-free H2O) using reagents from the Nextera DNA library Preparation Kit (Illumina #FC-121-103). Samples were then incubated at 37°C for 30min. DNA was isolated using a ZYMO Kit (#D4014). ATAC-seq libraries were first subjected to 5 cycles of pre-amplification. To determine the suitable number of cycles required for the second round of PCR the library was assessed by quantitative PCR as described in Buenrostro et al and the library was then PCR amplified for the appropriate number of cycles using Nextera primers. Samples were subject to a dual size selection (0.55x-1.5x) using SPRI beads (Beckman Coulter #B23317). Finally, the ATAC libraries were sequenced on a HiSeq 2500 platform on PE50 mode.