Homo sapiens
isolate
sALSnoFTLD4
age
88
biomaterial_provider
Prof. Janice Robertson, 60 Leonard Ave., 4KD481, Toronto, ON, M5T0S8
sex
female
tissue
brain, orbitofrontal cortex
disease
ALS with no FTLD
extracted_molecule
nuclear RNA
nuclei_isolation_procedure
Frozen orbitofrontal cortex (~50mg per sample) was dounce homogenized on ice in lysis buffer (0.32mM sucrose, 5mM CaCl2, 3mM Mg(Ac)2, 20mM Tris-HCl [pH 7.5], 0.1% Triton X-100, 0.5M EDTA [pH 8.0], 40U/mL RNase inhibitor in H2O), spun down at 800g for 10 min at 4C, then washed twice and resuspended in wash and resuspension buffer (1x PBS, 1% BSA, 0.2U/L RNase inhibitor). Resuspended nuclei were sorted by fluorescence activated cell sorting with DAPI (Roche) removing any debris and nuclei aggregates within DAPI-positive gating while aiming to capture ~6000 nuclei per sample. Library preparation was performed using either the 10X Chromium Single Cell 3 v2 or v3 platform following the manufacturer protocols. The QC of cDNA libraries was conducted on a 2100 Bioanalyzer (Agilent). The cDNA libraries were 100-bp paired end sequenced on either an Illumina NovaSeq 6000 SP XP or NovaSeq6000 S2 standard flow cell at the Princess Margaret Genomic Centre (Toronto, Ontario). Raw Illumina base call files from each sample were demultiplexed to produce FASTQ files with the cellranger mkfastq pipeline (10X Genomics). Reads were aligned to the pre-mRNA GRCh38-2020-A genome and quantified using cellranger count command on Cell Ranger v5.0.0 (10X Genomics).
data_processing
Fastq files were processed with CellRanger 5.0.0 count function with GRCh38-2020-A reference with the expected cells set to 6000 to produce the output filtered_feature_bc_matrix supplementary files.
BioSampleModel
Human