GSM1566012: 5fC_E14_PvuSealSeq_bio1_rep2; Mus musculus; OTHER
Genomic DNA was extracted with a Qiagen DNeasy Blood and Tissue Kit (Qiagen, Venlo, Limburg). To generate 5hmC library, 2µg of genomic DNA was digested with ~0.7 µg of PvuRts1I at room temperature for 2 hours. Next, 30 units of T4 Phage β-glucosyltransferase (New England Biolabs, Ipswich, MA) and 75µM UDP-6N3-Glc were added to the reaction and incubated at 37 oC for 2 hours. DNA ends created by PvuRts1I were ligated with 7µM Adapter P1 (top: ACACTCTTTCCCTACACGACGCTCTTCCGATCTNN and bottom: AGATCGGAAGAG CGTCGTGTAGGGAAAGAGTGT) with T4 DNA ligase (New England Biolabs) at 16 oC overnight. The next day, genomic DNA was sheared to around 200 bp by the Covaris s-series sonicator according to the suggested settings. The sheared genomic DNA was then purified with DNA Clean and Concentrator kit (Zymo, Irvine, CA). The purified DNA was reacted with 1 mM dibenzocyclooctyne-S-S-PEG3-biotin conjugate (Click Chemistry Tools, Scottsdale, AZ) at 37 °C for 2 hours. The DNA was then purified again with DNA Clean and Concentrator kit (Zymo). Subsequently, streptavidin beads (New England Biolabs) were added to the DNA and rotated at room temperature for 2 hours. The DNA captured on the beads was washed twice with 5mM Tris pH=8.0 and 1M NaCl. After that, the DNA was end repaired and dA-tailed with NEBNext® End Repair and NEBNext® dA-Tailing Module (New England Biolabs), respectively. Subsequently, 1µM adapter P2 (top: /5Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTC and bottom: GACTGGAGTTCAGACGTGTGCTCTTCCGATCT) was added to perform ligation with NEBNext® Ultra™ Ligation Module (New England Biolabs) at 20 °C for 15 minutes. Lastly, 100mM DTT was added to the reaction to cleave the disulfide bond in order to release the 5hmC library from the streptavidin beads. The 5hmC library was then amplified with NEB universal and index1 primer (New England Biolabs). The library DNA was purified via Ampure Beads (Beckman Coulter, Indianapolis, IN) with the ratio 1:1 and subject to next generation sequencing pipeline.For 5fC library construction, to ensure the complete conversion of 5hmC to 5gmC, 12 µg of E14 genomic DNA were first incubated with 180 units of T4-BGT and 450µM UDP-Glc at 37oC for 3 hours. Then fresh 180 units of T4-BGT and 450µM UDP-Glc were added to the reaction at 37oC for overnight. The next day, additional 180 units of T4-BGT and 450µM UDP-Glc were added to the reaction at 37oC for 3 hours. The genomic DNA was then purified via phenol chloroform extraction.  Subsequently, 100mM of NaBH4 was added to the DNA and incubated for 2 hours at room temperature. Genomic DNA was then precipitated by ethanol and ready for library construction.