GSM7677913: Embryonic stem cell, 5fC mapping by CLED-seq, rep1; Mus musculus; OTHER
The genomic DNA of mESCs was extracted using E.Z.N.A tissue DNA kit (Omega Bio-Tek Inc., Norcross, GA, USA) according to the manufacturer's recommended procedure. 10 μg of genomic DNA was used for the construction of sequencing library 10 μg of genomic DNA from mESCs was fragmented with a JY92-II N Ultrasonic Homogenizer to obtain 300–500 bp DNA fragments. The fragmented DNA was reacted with 5 μM biotin-ONH2 and 100 mM MES buffer (pH 5.0) at 37°C for 4 h. The resulting DNA was purified by ethanol precipitation to remove excess biotin-ONH2. The biotin-labeled DNA fragments was isolated by using PuriMag G-streptavidin magnetic beads  according to the manufacturer's recommended procedure. The mixture (50 μL) of enriched 5fC-containing DNA fragments and spike-ins were end-repaired and dA-tailed using Hieff NGS Fast-Pace End Repair/dA-Tailing Module kit according to the manufacturer's recommended protocol. As for the adapter ligation, 60 μL of end-repaired DNA and 5 μL of phosphorylated adapter (10 μM) were ligated using Hieff NGS Ultima DNA Ligation Module kit at 20°C for 6 h. The DNA was washed three times with water to remove the excess adaptor. Then 2 μL of proteinase K was added and incubated at 55°C for 30 min to release 5fC-containing DNA fragments. The resulting DNA was denatured by heating at 95°C for 10 min in 10% DMSO (v/v) solution and chilling in ice water. The deamination reaction was conducted in a 20-µL reaction mixture containing 20 ng DNA, 20 mM MES (pH 7.5), and 20 µM of A3A. The resulting DNA was subjected to PCR amplification by Q5U Hot Start High-Fidelity DNA polymerase. The PCR reaction (50 μL) included 10 µL of deaminated DNA, 10 μL of 5× Q5U reaction buffer, 1 µL of 10 mM dNTPs, 1 µL of Q5U Hot Start High-Fidelity DNA polymerase, 2.5 μL of pre-i7 primer (10 μM), 2.5 μL of pre-i5 primer (10 μM), and 23 μL of H2O. The amplification program started at 98°C for 30 s, followed by 10 cycles of 98°C for 10 s, 63°C for 1 min, 68°C for 1 min, and ended with 72°C for 10 min. PCR products were sequentially purified using 0.8× KAPA Pure beads followed by the second round of PCR amplification. The PCR reaction (50 µL) included 20 µL of purified DNA, 10 µL of 5× Q5 reaction buffer, 1 µL of 10 mM dNTPs, 1 µL of Q5 High-Fidelity DNA polymerase, 2.5 μL of i7 primer (10 μM), 2.5 μL of i5 primer (10 μM), and 13 μL of H2O. The amplification program started at 98°C for 30 s, followed by 8 cycles at 98°C for 30 s, 65°C for 1 min, 72°C for 2 min, and ended with 1 cycle at 72°C for 10 min. The PCR products were purified using 0.7× KAPA Pure beads. Library quality was assessed on the Agilent Bioanalyzer 2100 system. The library was sequenced on an Illumina platform and 150-bp paired-end reads were generated.