title: GSM8035808: Sequencing data for 5mC from HEK293T by BS-seq; Homo sapiens; Bisulfite-Seq
library_construction_protocol: Genomic DNA of HEk293T was extracted using Tissue DNA kit (Omega Bio-Tek Inc., Norcross, GA, USA) TAD-seq：The genomic DNA from HEK293T cells was isolated using the Tissue DNA kit (Omega Bio-Tek Inc., Norcross, GA, USA) following the manufacturer's protocol. Unmodified genomic DNA from lambda bacteriophage (Sangon Biotech, Shanghai, China) was added to the genomic DNA as a spike-in control (0.1% of spike-in DNA was added). The mixture was then sheared to an average size of 200-400 bp using a JY92-II N Ultrasonic Homogenizer (Scientz Biotechnology Co., Ltd, China). The fragmented DNA mixture was end-repaired and adenylated using the Hieff NGS Ultima Endprep Mix kit (Yeasen Biotechnology Co., Ltd, Shanghai). Subsequently, DNAm-adaptors (Table S5) were ligated to both ends of the repaired DNA using the Hieff NGS Ultima DNA Ligation Module kit (Yeasen, Shanghai, China), and the resulting DNA was purified using 0.9× KAPA Pure beads (Roche). Additionally, L-5mC-dsDNA was added as a spike-in to evaluate the deamination rates of C and 5mC (0.1% of L-5mC-dsDNA was added). The DNA mixture was then oxidized by nTET and purified using 0.9× KAPA Pure beads. Subsequently, the resulting DNA was denatured and deaminated using A3Am protein. The deaminated DNA was amplified by PCR with 6 cycles using 1 U of EpiMark Hot Start Taq DNA polymerase (New England Biolabs), 0.2 mM of dNTP, 0.4 μM of pre-P5 primer, and 0.4 μM of pre-P7 primer. Following purification using 0.9× KAPA Pure beads, the DNA products were amplified by PCR with 8 cycles using 1 U of Q5 Hot Start High-Fidelity DNA polymerase (New England Biolabs), 0.2 mM of dNTP, 0.4 μM of P5-index primer, and 0.4 μM of P7-index primer. The PCR products were purified with 0.9× KAPA Pure beads and examined using 2% agarose gel electrophoresis. The library quality was assessed on the Agilent Bioanalyzer 2100 system and then sequenced on an Illumina NovaSeq 6000 platform (Novogene Co., Ltd, Nanjing, China). BS-seq：The genomic DNA from HEK293T cells was isolated using the Tissue DNA kit (Omega Bio-Tek Inc., Norcross, GA, USA) following the manufacturer's protocol. Unmodified genomic DNA from lambda bacteriophage (Sangon Biotech, Shanghai, China) was added to the genomic DNA as a spike-in control (0.1% of spike-in DNA was added). The mixture was then sheared to an average size of 200-400 bp using a JY92-II N Ultrasonic Homogenizer (Scientz Biotechnology Co., Ltd, China). Next, ssDNA fragements were Bisulfite treated. After BS treatment, unmethylated Cytosine will change into Uracil, while methylated Cytosine will stay unchanged. Then, methylation sequencing adapters ligation, size selection and PCR amplification steps were performed to the DNA fragments. The library kit (Accel-NGS Methyl-Seq DNA Library Kit(Swift, USA) adds a low complexity polynucleotide tail with an average length of 8 bases to the 3' end of each fragment during the addition of the first NGS adapter molecule. If these tails are not trimmed bioinformatically from the sequencing data, it is normal and expected to observe them at the beginning of Read 2 (R2). When read length is close to fragment size, the tail may also be observed toward the end of Read 1 (R1) data. Therebefore, it's highly recommended to trim 10 bases from the beginning of both R1 and R2 to eliminate any artifactual cytosine methylation introduced as a result of filling inoverhangs during end repair steps of conventional dsDNA library preparation and low quality bases due to bisulfite treatment. Subsequently, library qualitywas assessed on the Agilent 5400 system（Agilent，USA） and quantified by QPCR (1.5 nM). Pair-end sequencing of sample was performed on an Illumina NovaSeq 6000 platform (Illumina, USA). whole-genomic sequencing by TAD-seq and BS-seq