title: GSM5100470: 5hmC SEAL-seq rep1; Homo sapiens; OTHER
library_construction_protocol: 5hmC-Seal was performed as previously described with modifications. Briefly, 80 μg genomic DNA was resuspended in TE buffer and sonicated to 200 bp fragments using a Covaris S220 sonicator. Fragmented DNA was precipitated by ethanol and sodium acetate and resuspended in TE buffer. The selective 5hmC chemical labeling was performed in 100 μl glucosylation buffer (50 mM HEPES buffer pH 8.0, 25 mM MgCl2) containing above fragmented DNA, β-GT (NEB, Cat. No. M0357), UDP-Azide-Glucose (Active Motif, Cat. No. 55020), and incubated at 37°C for 1.5 hr. After the reaction, DNA was cleaned up with QIAquick Nucleotide Removal Kit. The labeled DNA was eluted with ddH2O, after which 1mM DBCO-PEG4-Biotin (Click Chemistry Tools) was added and incubated at 37°C for 2 hours. Then, the biotin-labeled DNA was pulled down by C1 Streptavidin beads (ThermoFisher, Cat. No. 650-01) for 30 minutes at room temperature. The captured DNA fragments were processed for library construction as described in SAR-seq (above).  For 5fC-SEAL, we modified the previously described protocol, which reduces 5fC to 5hmC using NaBH452. We labeled the newly generated 5hmC (derived from 5fC reduction with NaBH4) with an azide-modified glucose as described above for 5hmC-Seal. In brief, 80 μg of fragmented i3Neurons genomic DNA was incubated in 100 μl glucosylation buffer (50 mM HEPES buffer pH 8.0, 25 mM MgCl2) containing unmodified UDP-Glucose (NEB, Cat No. M0357), and β-GT for 1.5 hours at 37°C. The labeled DNA was cleaned up with QIAquick Nucleotide Removal Kit. Then, an equal volume of freshly prepared NaBH4 (Aldrich, Cat. No. 213462) solution was added to the glucose-blocked DNA solution. The reaction mixture was vortexed and incubated in a Thermomixer for 15 minutes at room temperature. The chemical labeling and capture were performed as described above for 5hmC-SEAL.