Sox2+ FACS sorted cells from caudal ends of WT embryos
strain
C57BL/6N
developmental stage
TS12
Sequenced DNA Library
library_strategy
ATAC-seq
library_source
GENOMIC
library_selection
other
library_construction_protocol
In total, 1,000-5,000 cells were used per ATAC-Seq experiment. After spinning down the FACS sorted cells, the pellets were resuspended in 50ul of lysis buffer (10mM Tris pH7.4, 10mM NaCl, 3mM MgCl2, 0.1% NP-40) and immediately spun down at 500g and 4°C for 10 min. The supernatant was discarded and each pellet was resuspended in the transposition reaction mix (25ul 2x TD buffer, 2.5ul Tn5 transposase, 22ul H2O) and incubated at 37°C for 30 minutes. After incubation, the reaction was stopped with the addition of PB buffer (Qiagen) and the tagmented DNA was purified using the MinElute kit (Qiagen). The DNA was combined with the Nextera PCR primers and 2x Kapa HiFi Hotstart Readymix and pre-amplified (98°C 30 seconds, 8x [98°C 10 seconds, 63°C 30 seconds, 72°C 1 minute] in a 50ul volume. To determine the remaining cycles to avoid potential overamplification, 5ul of the preamplification mix was combined with the primers, 1x Evagreen Sybr green (Jena Biosciences) and 2x Kapa HiFi Hotstart Readymix in a 15ul total volume and run for 30 cycles on a StepOne Plus. The remaining 45ul pre-amplified samples were amplified for a further 9 cycles, resulting in a total of 17 cycles. The libraries were purified using MiElute column (Qiagen) and the concentration was measured using the DNA HS Qubit assay (life). Approximately 4ng of each library were run on a DNA HS Bioanalyzer ChIP to verify library size and calculate molarities.