title: GSM5818579: G2M_EMseq_rep1; Mus musculus; OTHER
library_construction_protocol: Cultured ES cells were EdU labelled, harvested, and fixed in 70% ethanol. Cells were treated with Click-iT to label EdU+ cells and PI for DNA content and sorted via fluorescence-activated cell sorting to obtain pure G1 and G2/M populations. Genomic DNA was isolated from sorted cells using the Zymo Duet MiniPrep kit (D7001). Libraries were generated using the EM-seq method (NEB, E7120S). 125 ng DNA per sample was mixed with 1 pg pUC19 DNA and 20 pg lambda DNA and sonicated to 200 bp using a Covaris E220. Drosophila DNA was sonicated in parallel, 7.5 pg was added to sonicates, and PCR-grade water was added to a final volume of 50 μL. 7 μL NEBNext Ultra II End Prep Reaction Buffer and 3 μL NEBNext Ultra II End Prep Enzyme Mix was added to each sample, mixed, and incubated at 20 °C for 30 minutes followed by 65 °C for 30 minutes in a thermocycler with a lid heated to 65 °C. On ice, 2.5 μL NEBNext EM-seq Adaptor, 1 μL NEBNext Ligation Enhancer, and 30 μL NEBNext Ultra II Ligation Master Mix was added to each sample, mixed, and incubated at 20 °C for 60 minutes in a thermocycler with an opened lid. Samples were purified using NEBNext Sample Purification Beads, 1.18:1 bead:sample ratio, and eluted in 28 μL Elution Buffer. 10 μL freshly prepared TET2 Reaction Buffer, 1 μL Oxidation Supplement, 1 μL DTT, 1 μL Oxidation Enhancer, and 4 μL TET2 enzyme were added to each sample and mixed. 5 μL freshly diluted 400 μM Fe(II) Solution was added to each sample, mixed, and incubated at 37 °C for 60 minutes in a thermocycler with a lid heated to 45 °C. On ice, 1 μL Stop Reagent was added to each sample, mixed, and incubated at 37 °C for 30 minutes in a thermocycler with a lid heated to 45 °C. Samples were purified using NEBNext Sample Purification Beads, 1.8:1 bead:sample ratio, and eluted in 16 μL Elution Buffer. Samples were denatured by adding 4 μL freshly prepared, mixing, and incubating at 50 °C for 10 minutes in a thermocycler with a lid heated to 60 °C. On ice, 68 μL PCR-grade water, 10 μL APOBEC Reaction Buffer, 1 μL BSA, and 1 μL APOBEC enzyme were added to each sample, mixed, and incubated at 37 °C for 3 hours in a thermocycler with a lid heated to 45 °C. Samples were purified using NEBNext Sample Purification Beads, 1:1 bead:sample ratio, and eluted in 20 μL Elution Buffer. Libraries were amplified by adding 5 μL EM-seq Index Primer and 25 μL NEBNext Q5U Master Mix to each sample, mixing, and cycling with the following conditions: 98 °C, 30 seconds; 5 cycles of: 98 °C, 10 seconds; 62 °C, 30 seconds; 65 °C, 60 seconds; followed by a 65 °C, 5 minutes final extension. Libraries were purified in two rounds using NEBNext Sample Purification Beads, first round 0.9:1 bead:sample ratio; second round 1:1 bead:sample ratio, and eluted in 10 μL Elution Buffer. Libraries were checked for quality control by BioAnalyzer and Qubit quantification before sequencing.