title: GSM9628958: EpCAM+, DN3 cells 2; Mus musculus; ATAC-seq
library_construction_protocol: DN3 cells were first gained by flow sorting on BD-FACSAria Fusion. Then, EpCAM-based separation using biotin-conjugated anti-EpCAM antibody and anti-biotin microbeads, according to the manufacturer's protocol (Miltenyi Biotec) on Miltenyi autoMACS® Pro Separator (130-092-545)，where EpCAM⁺ cells were positively selected while the unlabeled fraction constituted EpCAM⁻ cells. Two cell group were then lysised for the ATAC-seq, following DNA fragmentation, purification, amplification, the DNA products were sequencing by HaploX. Sequencing libraries were constructed using the Vazyme TruePrep DNA Library Prep Kit according to the manufacturer's instructions for the appropriate input DNA amount (50 ng, 5 ng, or 1 ng). The protocol is based on a tagmentation reaction. Briefly, the starting DNA was mixed with TruePrep Tagment Enzyme Mix (TTE Mix), which contains a transposase and two equimolar adapters (Adapter 1 and Adapter 2), in the presence of 5× TTBL buffer. The mixture was incubated at 55°C for 10 minutes to achieve simultaneous DNA fragmentation and adapter ligation. The tagmentation reaction was immediately stopped by adding 5× TS buffer and incubating at room temperature for 5 minutes. The fragmented DNA was then purified using VAHTS DNA Clean Beads (required for the 50 ng input protocol) or used directly for PCR enrichment. The PCR enrichment reaction was set up with the purified/stopped DNA, 5× TAB buffer, PCR Primer Mix (PPM), N5XX and N7XX indexing primers, and TAE polymerase. Thermal cycling was performed as follows: 72°C for 3 minutes for strand displacement; followed by a cycle of 98°C for 30 seconds; and then 5-15 cycles of 98°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds; with a final extension at 72°C for 5 minutes. The number of amplification cycles was optimized based on the starting DNA amount (5-9 cycles for 50 ng, 8-12 cycles for 5 ng, and 11-15 cycles for 1 ng). The amplified products were subjected to a two-round size selection and purification using VAHTS DNA Clean Beads. The volumes of beads used in each round were adjusted according to the starting DNA amount to enrich for fragments of the desired length (e.g., for 50 ng input, 0.60× and 0.15× beads were used to select a 300-700 bp library). The final purified library was quality-controlled and stored at -20°C prior to sequencing.
identifiers.namespace: GEO
identifiers.id: GSM9628958_r1