GSM9814870: Pooled snRNAseq samples; Mus musculus; RNA-Seq
At 2 months of age (post-natal day 58), mice were cervically dislocated. Brains were kept on ice and harvested into cold 1X PBS. The cortex was microdissected under the microscope isolated and flash frozen in liquid nitrogen. Nuclei isolation was performed by Princess Margaret Genomics Centre (PMGC). Nuclei extraction was performed on 54.5 - 64.6mg of flash frozen tis-sue per brain as per the manufacturer's instructions (Nuclei Isolation Kit, CG000505, 10x Ge-nomics) until step l on page 31, after which suspended in nuclei separation buffer and anti-nucleus microbeads (Miltenyi, 130-132-997). Isolated nuclei were processed using the GEM-X Flex Sam-ple Preparation v2 Kit (PN-1000781). For each reaction, 1.96-2.44x106 nuclei were recovered. To perform the Flex assay, we used Chromium GEM-X Flex Gene Expression protocol (CG000780, 10X Genomics). 10,000 nuclei were targeted per sample. Samples were pooled in equal amounts based on nuclei counts prior to loading on the Chromium. For Flex, samples were pooled prior to library prep. Custom probes were used to identify zsGreen and iCre. Probe sequences: iCre_1 AGGATCTGCACACAGACAGGAGCATCTTCCAGGTGTGTTCAGAGAAGGCC; iCre_2 CTCTGATTCTGGCAATTTCGGCAATGCGCAGCAGGGTGTTGTAGGCAATG; iCre_3 CAGAACAGGTAGTTGTTGGGGTCATCAGCCACACCAGACACAGAGATCCA. Probe sequences: zsGreen_1 GCGGGGCAGGAGTTCTTGAAGTAGTCGACGATGTCCTGGGGG-TACTCGGT; zsGreen_2 CGTAGAACTTGGACTCGTGGTACATGCAGTTCTCCTCCAC-GCTCACGGTG; zsGreen_3 GGGATGATCTTCTCGCAGGAGGGCTCCCAGTT-GTCGGTCATCTTCTTCAT. Library preparation was performed using the GEM-X Flex GEM & Library Kit, 4 rxns (PN-1000782). Equal molar amounts of each library for all samples were pooled and sequenced at an expected depth of 35,000 reads/nuclei using the Illumina Novaseq X 100 cycles sequencing system (Princess Margaret Centre for Research and Genomics). Nuclei were pooled prior to library preparation (Pool_135) and sequenced as a single library. Cells were computationally demultiplexed into N1, N2, and N3 .All samples share the same raw FASTQ files.