title: GSM4550957: Dnmt1 CR embryo group 1; Mus musculus; MBD-Seq
library_construction_protocol: A pair of single-guide RNAs (sgRNAs) were designed to target exon-32 of mouse Dnmt1 gene and were synthesized (see Supplementary_Data_File_S1 for sgRNA information; Macrogen, Korea). Both sgRNAs (40 ng each) and Cas9 recombinant protein (40 ng; Toolgen, Korea) were mixed and incubated  in a microinjection buffer (0.1 mM EDTA, 10 mM Tris-Cl, pH 7.4) for 15 min at 37°C (1). Microinjection was performed 24 hrs after human chorionic gonadotropin injection. To visualize the pronuclei, cumulus-free zygotes were centrifuged at 13,000 rpm for 5 min, injected with the cocktail of sgRNA and Cas9, and cultured in M16 media for 48 hrs. Microinjection was performed under an inverted microscope equipped with micromanipulator and a microinjector (Leica). The resulting 4- and 8-cell stage embryos were treated with Tyrode's solution (Sigma) for 2 minutes to remove zona pellucida, washed 3 times with 0.1% PVA-PBS, and collected for either genotyping or MBD-sequencing. MBD-seq libraries were prepared using MethylMiner Methylated DNA Enrichment Kit (Thermo) according to manufacturer's protocol
attributes.tag: GEO Accession
attributes.value: GSM4550957