title: GSM7528345: Sequencing data for 5hmC from human normal lung tissue by ACE-seq; Homo sapiens; OTHER
library_construction_protocol: Genomic DNA of human normal lung tissue was extracted using Tissue DNA kit (Omega Bio-Tek Inc., Norcross, GA, USA) SSD-seq：Genomic DNA of human normal lung tissue was extracted using Tissue DNA kit (Omega Bio-Tek Inc., Norcross, GA, USA) according to the manufacturer's recommended procedure. The unmodified genomic DNA of lambda bacteriophage (Sangon Biotech, Shanghai, China) was added to the genomic DNA of human normal lung tissue as spike-in control (0.1% of spike-in DNA was added). The mixture was sheared to an average size of 250-400 bp using a JY92-II N Ultrasonic Homogenizer (Scientz Biotechnology Co., Ltd, China). The resulting fragmented DNA was end-repaired and adenylated using a Hieff NGS Ultima Endprep Mix kit (Yeasen Biotechnology Co., Ltd, Shanghai). Then, SSD-adaptor was ligated to both ends of repaired DNA using a Hieff NGS Ultima DNA Ligation Module kit (Yeasen) and the resulting DNA was purified using 0.8× KAPA Pure beads (Roche). To the resulting mixture, DNA-L-5mC and DNA-5hmC were added as spike-ins to evaluate the deamination rates of 5mC and 5hmC (0.1% of DNA-L-5mC and 0.1% DNA-5hmC were added). The DNA mixture was denatured followed by deamination using eA3A-v10. The deaminated DNA was amplificated by PCR with 5 cycles using pre-P5 primer, pre-P7 primer (Table S4) and Q5U Hot Start High-Fidelity DNA polymerase (New England Biolabs). After purification using 0.8× KAPA Pure beads, DNA products were then amplificated by PCR with 10 cycles using P5-index primer, P7-index primer and Q5 Hot Start High-Fidelity DNA polymerase (New England Biolabs). The PCR products were purified with 0.8× KAPA Pure beads and examined using 1.5% agarose gel electrophoresis.