GSM5766257: HEK293_DRB_2h_release_30min_spKAS-seq.rep2; Homo sapiens; OTHER
After labeling, cells were harvested for genomic DNA isolation by using the PureLink Genomic DNA mini kit (Thermo, K182002). Around 2 µg genomic DNA was then diluted in 100 µL 1× PBS supplemented with 25 mM K3BO3 and 20 mM DBCO-PEG4-biotin (Sigma, 760749). The mixture was gently shacked at 37 °C for 1.5 h, after which 5 µL RNase A (Thermo, 12091039) was added, followed by another 5 min incubation at 37 °C. DNA was then purified from the reaction by using the DNA Clean & Concentrator-5 kit (Zymo, D4013) and was fragmented to 150-350 bp by sonicating 30 cycles at 30s-on/30s-off setting using Diagenode Bioruptor Pico. Save 5% of the sonicated DNA as input and use the rest for enrichment with 10 µL Dynabeads MyOne Streptavidin C1 (Thermo, 65001). The beads were washed by 1× B&W buffer (5 mM Tris-HCl pH 7.4, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween-20), resuspended in 95 µL 2× B&W buffer, and mixed with sonicated DNA. The binding was performed at room temperature for 15 min. The beads were washed once in 1× B&W buffer and then washed twice with 100 mM NaOH solution to denature the dsDNA and remove the DNA strands that are not labeled by N3-kethoxal. A final wash with 1× B&W buffer was followed, and DNA was eluted in 10 µL H2O by heating the beads at 95 °C for 10 min. Input and enriched DNA were used for library construction by using the Accel-NGS Methyl-seq DNA library kit (Swift, 30024). Libraries were sequenced on Illumina platforms with pair-end more with around 60 million reads per library.