GSM9793427: Hnf1a_Scrambled_sgRNA-Rep1; Mus musculus; OTHER
Collect 15 mouse oocytes Whole-genome bisulfite sequencing (WGBS) was performed on mouse oocytes using 15 oocytes as input, whereas Enzymatic Methyl-seq (EM-seq) was applied to human oocytes starting from a single cell due to its high efficiency with minimal input. Library preparation and sequencing: Mouse metaphase II (MII) oocytes (n = 15 per replicate, two biological replicates per group) were used for WGBS. Briefly, cells were lysed in buffer (10 mM Tris-HCl, pH 7.4, 2% SDS), and genomic DNA was extracted and digested with proteinase K. Purified DNA was subjected to bisulfite conversion using the EZ DNA Methylation-Gold Kit (ZYMO, D5008), followed by purification with Agencourt AMPure XP beads (Beckman). The converted DNA was end-repaired and dA-tailed, then ligated to Illumina sequencing adapters. Libraries were amplified for 13 cycles using Illumina universal PCR primer and indexed primers. For human samples, individual oocytes were lysed and genomic DNA were fragmented to ~ 300 bp using a DNA fragmentation device (Shanghai Jingxin, DNA Fragmentation Model 26A). Libraries were constructed using the NEBNext® Enzymatic Methyl-seq kit (NEB, E7120 L) and the Unique Dual Index Primer pairs (NEB, E7140L). Unmethylated lambda phage DNA and CpG-methylated plasmid pUC19 DNA were included as negative and positive controls. For single-oocyte libraries, amplification was performed for 18 cycles. Libraries were purified using the DNA Clean & Concentrator-5 kit (ZYMO, D4013). Finally, equimolar amounts of libraries were pooled and subjected to paired-end sequencing on an Illumina NovaSeq 6000 platform.