GSM9536665: mESC PEM-seq NanogSE locus; Mus musculus; OTHER
PEM-seq was performed following the published protocol with minor modifications. Two million GFP-positive viable cells were isolated by FACS, and genomic DNA was extracted using standard procedures. Purified genomic DNA was used as input for downstream shearing and primer-extension workflow. Purified DNA was sheared to a peak size of 300-700 bp using an M220 focused ultrasonicator (Covaris). For primer extension, biotinylated primers were subjected to repeated cycles of annealing and denaturation with 20 µg of fragmented genomic DNA, followed by primer extension using Bst Polymerase 3.0 (M0374S, NEB). Excess biotinylated primers were removed using VAHTS DNA Clean Beads (1.2:1 bead-to-sample ratio). The purified DNA was denatured at 95°C for 5 minutes and immediately chilled on ice for 5 minutes. Biotinylated extension products were captured with Dynabeads MyOne Streptavidin C1 and washed with B&W buffer and nuclease-free water according to the manufacturer's instructions. For bridge-adapter ligation, beads were resuspended in a ligation mixture containing 15% PEG8000, T4 DNA ligase (EL0011, Thermo Scientific), and annealed bridge adapters, and incubated overnight at room temperature. Bead-bound ligation products were washed and subjected to on-beads nested PCR using I5 nested primers, I7 index primers, and Taq polymerase (TransGen Biotech) for 16 cycles. PCR products were purified using VAHTS DNA Clean Beads and subsequently amplified with FastPfu (TransGen Biotech) to incorporate Illumina P5 and P7 adapters. Final PEM-seq libraries were sequenced on a DNBSEQ-T7 platform.