title: GSM8524036: DKO-Fx_1_5hmC; Mus musculus; OTHER
library_construction_protocol: The collected spermatozoa were centrifuged for 3 min at 1100g and resuspended in 200 μl of solution A (75 mM NaCl and 25 mM EDTA) and 200 μl of solution B (10 mM Tris-HCl pH 8, 10 mM EDTA, 2% SDS, 80 mM DTT) complemented with 1 μg of RNAse A (Qiagen) and incubated for 1 h at 37°C. One hundred μg of Proteinase K (Sigma-Aldrich) per 0.5 ml of solution A+B was subsequently added and incubated overnight at 55°C. Sperm genomic DNA was further purified using phe-nol:chloroform:isoamyl alcohol extraction followed by ethanol precipitation. DNA pellet was resuspended in RNAse/DNAse free distilled water (Invitrogen). For EM-seq: A total of 200 ng of genomic DNA was combined with 1.0 ng of unmethylated Lambda DNA (New England Biolabs) and 0.1 ng of CpG methylated pUC19 (New England Bi-olabs). This mixture was then subjected to digestion using 400 units of MspI (New England Biolabs) in NEB Ultra II buffer (New England Biolabs) in a final volume of 57 μL. The digestion was carried out for 2 hours at 37°C. Following enzymatic digestion, the resulting DNA fragments underwent end-repair and A-tailing using 15 units of Klenow 3'-5' exo- (New England Biolabs). Once end-repair and A-tailing were com-pleted, Illumina universal methylated adapters were ligated to the DNA fragments using the NEB Ultra II ligation module, following the manufacturer's recommenda-tions. After ligation, the DNA fragments were purified and size-selected using a two-step SPRI beads purification method to retain fragments ranging from 40 bp to 400 bp. The cleaned ligated DNA was eluted in 10 mM Tris pH 8. The purified product was then converted using the Enzymatic methyl-seq module (New England Biolabs), according to the manufacturer's instructions. In brief, 5mC were converted to 5hmC using TET2, and subsequently, 5hmC were protected from APOBEC deamination through T4-BGT glycosylation. After glycosylation, libraries were denatured at 85°C for 10 minutes in the presence of 20% formamide. Unmodified cytosines were then subjected to APOBEC deamination. The converted product underwent PCR amplifica-tion using indexing primers and KAPA HiFi Hotstart U+ Ready Mix 2X polymerase in a final volume of 50 μL for 9 cycles. The resulting PCR product was purified using a 1.2X SPRI ratio and subsequently size-assessed using capillary electrophoresis on a Fragment Analyzer.                                                                                                         For EhM-seq: A starting input of 200 ng DNA was used, along with 1.0 ng of unmethylated Lambda DNA and 0.1 ng of CpG methylated pUC19. After MspI digestion and Klenow 3'-5' exo- mediated repair, pyrrolo-dC Illumina adapters were utilized in the ligation step instead of 5mC adapters. During the conversion step involving TET2, H2O replaces TET2 enzyme and Fe2+ to prevent 5mC protection. The ensuing product was deami-nated, amplified, assessed, and sequenced under conditions identical to those of RREMseq libraries.
identifiers.namespace: GEO
identifiers.id: GSM8524036_r1