RRBS of Mus musculus: hippocampus APP_WT genotype
After the completion of behavioral testing, mice were euthanized via cervical dislocation and brain tissue was dissected. Hippocampi randomly selected from 15 female mice (AppNL-G-F and AppNL-F: n=5 mice; WT: n=4 mice) were used to perform DNA methylation analysis. The DNA was extracted and purified using Zymo Genomic DNA Clean & Concentrator-10 kit (Cat.N D410, Irvine, CA). Subsequently, purity of the DNA was verified using Qubit (ThermoFischer, Waltham, MA) and NanoDrop (ThermoFischer, Waltham, MA) analyses. Approximately 80 ng genomic DNA was digested for 2 h with MspI restriction enzyme (New England Biolabs, Ipswich, MA), which cuts the DNA at every occurrence of the CCGG sequence, resulting in fragments starting with a CpG site. Libraries were prepared with the NEBNext Ultra II Modules (New England Biolabs) and the NEBNext Methylated Adaptor (New England Biolabs). The ligated DNA was size-selected using Sera-Mag Select magnetic beads (GE Healthcare Life Sciences, Chicago, IL) to produce a final library size of~350 bp. Bisulfite conversion was performed with the EZ DNA Methylation-Gold Kit (Zymo Research, Irvine, CA), followed by PCR amplification with the NEBNext Q5U polymerase and NEBNext Multiplex Oligos for Illumina (New England Biolabs) to barcode each library. The resulting libraries were normalized and multiplexed for single-end (SE X75) sequencing on the Illumina NextSeq 500 platform at the Massively Parallel Sequencing Shared Resource (MSRP) at OHSU.