title: GSM8543938: d1A38, d1A39, mESC, Mouse; Mus musculus; OTHER
library_construction_protocol: • Cell lines We utilized a female mouse embryonic stem cell (mESC) line derived from blastocysts at embryonic day 3.5 (E3.5) from crossbred embryos (female C57BL/6J × male CAST/EiJ). The procedures for derivation and culturing followed the methods provided by Arenas et al. (2006). • Cell culture Cells were cultured on flasks coated with 0.1% gelatin in an ES cell medium containing serum. The medium composition included DMEM (Gibco, 10829018), 15% ESC-grade fetal bovine serum (FBS) (VivaCell, C04002-100), 0.1 mM β-mercaptoethanol (Gibco, 21985023), 1% P/S (ThermoFisher, 10378016), NEAA (Sigma, M7145), L-Glutamine (Gibco, 25030-081), nucleotides (Millipore, ES-008-D), and 1000 U/ml leukemia inhibitory factor (LIF) (PeproTech, AF-250-02-25UG). Additionally, the medium was supplemented with 2i: 3 μM Gsk3 inhibitor CT99021 (Selleck, S1263) and 1 μM MEK inhibitor PD0325901 (Selleck, S1036).  • Cell differentiation Differentiation was initiated by withdrawing 2i/LIF in DMEM supplemented with 10% FBS, 1% P/S, NEAA, L-Glutamine, nucleotides, and 0.1 mM β-mercaptoethanol. The cells were plated at a density of 1.5 × 10⁴ cells/cm² on 6 well cell culture plates coated with 10 μg/ml fibronectin (GLPBIO, GP23499). The single-cell preamplification product was diluted to 5ng/ul and 2 μL of each diluted sample was transposed by the addition of 3 μL Transposition Mixture (1 μL 5 X TTBL, 0.25 μL TTE Mix V50 (Vayme TD501), 1.75 μL water) and incubated at 55 °C for 10 min. Transposition was stopped by adding 1.25 μL 0.2% SDS and then the plate 40 was incubated at room temperature for 10 min. For a slight enrichment of cDNA reads, 2 μL 5 μM i5 unique dual (UD) index and 11.75 ul PCR mixture (4 μL 5X KAPA HiFi GC Buffer, 0.6 μL 10 mM dNTP, 0.2 μL 50 μM RNA-P7 primer, 0.4 μL KAPA HiFi DNA Polymerase (KAPA KK2102) and 6.55 μL water) were added to each well and incubated with the following program: Step 1: 72 °C × 3 min; Step 2: 98 °C × 30 s; Step 3: 98 °C × 15 s, 60 °C × 30 s, 72 °C