title: GSM8865975: GAD2_APP_2; Mus musculus; RNA-Seq
library_name: GSM8865975
library_construction_protocol: Mouse brain cortex tissues (one hemisphere of the cortex for each animal) were collected and put into 1.5ml RNAase free Eppendorf tubes, flash-frozen using liquid nitrogen, and stored at –80 ̊C. For each sample, we pooled 2 mouse brain cortex tissues into one tube for Single-nucleus suspensions. Single-nucleus suspensions were prepared following the protocol we described previously (McLaughlin et al., 2022). Nuclei were stained by Hoechst-33342 (1:1000; >5min). Next, we collected nuclei using Fluorescence activated cell sorting (FACS) to remove debris. We used the BD Aria III sorter for collecting nuclei. Hoechst+ nuclei were collected during sorting into a 1.5ml tube with 200ul 1x PBS with 0.5% BSA as the receiving buffer (RNase inhibitor added). For each 10x Genomics run, 80k–150k nuclei were collected. Nuclei were spun down for 10 min at 950g at 4 ̊C, and then resuspended using 40ul of 1x PBS with 0.5% BSA (RNase inhibitor added). 2ul nucleus suspension was used for counting the nuclei with hemocytometers to calculate the concentration. 20K nuclei were loaded into the 10X chip for each capture to target 10k nuclei for each run. 10x Genomics sequencing libraries were prepared following the standard protocol from Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (Dual Index) with following settings. All PCR reactions were performed using the Biorad C1000 Touch Thermal cycler with 96-Deep Well Reaction Module. Cycle numbers were used as the 10x protocol recommended for cDNA amplification and sample index PCR. As per 10x protocol, 1:10 dilutions of amplified cDNA and final libraries were evaluated on Agilent TapeStation. The final library was sent to Novogene Corporation Inc. for Illumina NovaSeq PE150 S4 lane sequencing with the dual index configuration Read 1 28 cycles, Index 1 (i7) 10 cycles, Index 2 (i5) 10 cycles, and Read 2 90 cycles. A PhiX control library was spiked in at 1% concentration. The sequencing depth is about 43-60K reads per nucleus.
identifiers.namespace: GEO
identifiers.id: GSM8865975_r1