title: GSM8965003: Multiome, WT mice, ENT, 2 months, ATAC; Mus musculus; ATAC-seq
library_name: GSM8965003
library_construction_protocol: From 5xFAD or wild-type mice, both on the C57BL/6 background, from 2 months or 8 months of age, animals were anesthetized under isoflurane induction and underwent cardiac perfusion with cold 1xPBS to remove blood from the brain. Brain tissue was immediately extracted, and the entorhinal cortex was freshly dissected out of each sample. Single-nucleus ATAC-seq and RNA-seq were conducted using the 10x Multiome kit. To isolate the nuclei, tissue was homogenized using Lysis Buffer and manually disrupted using pestles, followed by a 4-minute incubation period. Samples were centrifuged at 500g for 5 minutes at 4°C, and the supernatant was discarded. Subsequently, 1 ml of Nuclei Wash and Resuspension buffer (NWR) was added and incubated for 5 minutes. The pellet was carefully resuspended using a P1000 pipette and centrifuged again at 500g for 5 minutes at 4°C. This wash-resuspension-centrifugation process was repeated twice more. After the third centrifugation, the supernatant was discarded, and 200μL of NWR buffer + 20μL 7AAD (1:100) was added and passed through a 40μm filter directly into a tube used for flow cytometry sorting. Cells were sorted at the UC Irvine Institute for Immunology Flow Cytometry Facility to remove debris and distinguish viable nuclei. Following sorting, the samples underwent a permeabilization process, which began with centrifugation at 500g for 5 minutes at 4°C. Subsequently, 100μL of 0.1X Lysis buffer was added, followed by resuspension and a 2-minute incubation. Next, 1mL of Washing Buffer was added, and the sample was centrifuged again for 5 minutes under the same conditions. Then, 500μL of Diluted Nuclei Buffer was added and carefully mixed with a pipette. Finally, the resulting sample was spun down and submitted to the UC Irvine's Genomics Research and Technology Hub for further processing.