title: GSM3656321: 12ng 5hmc Jump-seq rep2; Mus musculus; OTHER
library_construction_protocol: mESCs genomic DNA was purified using Quick-DNA™ Microprep Plus Kit (Zymo Research). The Tn5 reaction protocol has been described previously1. In brief, genomic DNA was fragmented and tagmented by mixing 4 µL 5 × TAPS-MgCl2-PEG 8,000 (for DNA < 1 ng) or 5 × TAPS-MgCl2-DMF, 1 µL of the assembled Tn5 and adaptor complex, genomic DNA and suitable volume of H2O up to 20 µL. Buffer used were: TAPS-MgCl2-PEG 8,000 (50 mM TAPS-NaOH, pH 8.5, 25 mM MgCl2, 40% PEG 8,000) or 5 × TAPS-MgCl2-DMF (50 mM TAPS-NaOH, pH 8.5, 25 mM MgCl2, 50% DMF). For 48 ng genomic DNA, 1 µL of 2-fold diluted Tn5 is sufficient; for 24 ng genomic DNA, Tn5 needs to be diluted 4 folds and for less genomic DNA, Tn5 should be diluted accordingly. For 600 pg genomic DNA or 100 cells, Tn5 should be diluted 320 folds and added 1 µL into the reaction. The reactions were incubated for 7 min at 55℃, and Tn5 was inactivated for 7 min at 55℃ after adding 1 µL proteinase K (Thermo Fisher Scientific). Typically, all the genomic DNA was converted to fragments of average size 200-300 bp. The fragmented DNA was purified by DNA Clean & Concentrator™-5 kit (Zymo Research) and eluted into H2O. T4 bacteriophage β-GT and Tet was prepared as previously described3,4. The 5-hmC labeling reactions were performed in a 20 μL solution containing 50 mM HEPES buffer (pH 8.0), 25 mM MgCl2, tagmented genomic DNA (100-500 bp), 100 μM UDP-6-N3-Glu, and 1 μM wild-type β-GT. The reactions were incubated for 1 h at 37 °C. In the case of 5mC, the strategy of coupling Tet oxidation with β-GT was used5: 5hmC base is protected by transferring a regular glucose from UDP-glucose using β-GT, followed by purifica-tion with DNA Clean & Concentrator™-5 kit (Zymo Research). After the azide-glucose transfer reaction, the DNA substrates were purified by Clean & Concentrator™-5 kit (Zymo Research) and reconstituted in H2O. The click chemistry was performed with addi-tion of 0.8 nmol dibenzocyclooctyne modified Jump P5 probe into the DNA solution, and the reaction mixture was incubated for 24 h at 37 °C. The click products were incubated with Dynabeads™ MyOne™ Streptavidin C1 (Thermo Fisher Scientific) in 1 × B & W Buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 2 M NaCl) at RT for 20min (the click reactions with different barcoded Jump P5 probe could be pooled together for purification), followed by extensively washing steps: 1 × B & W Buffer 1 time, 0.15 M NaOH 1 time, 0.1 M NaOH twice, 1 × B & W Buffer with 200M Tris-HCl pH 7.5 one time, H2O three times and finally the beads were resuspended in 6 µL H2O and 2 µL of 5 × Sequenase Reaction Buffer for annealing (the loop shape Jump probe formed hairpin to initiate primer ex-tension). The reaction was heated at 95 °C and slowly ramped to 4 °C in Thermocycler. 1 µL Sequenase Version 2.0 DNA Polymer-ase (Thermo Fisher Scientific) and 1 µL dNTP (NEB) were added in the annealed solution, incubated at 37 °C for 2 h. Then 1 µL USER® Enzyme (NEB) was added to cleave at the dU linker region to release the newly synthesized strand. The 11 µL Jump prod-ucts were subjected to library construction with limited cycle amplification. Next-generation sequencing (NGS) libraries were gen-erated using NEBNext® Ultra™ II Q5® Master Mix (NEB), NEBNext® Multiplex Oligos for Illumina® (NEB) for NEB kit compat-ible Jump P7 adaptor and Jump P5 probe and Illumina Nextera DNA oligo sets for Nextera DNA kit compatible Jump P7 adaptor and Jump P5 probe.