Homo sapiens
isolate
CTRL2
age
59
biomaterial_provider
Prof. Janice Robertson, 60 Leonard Ave., 4KD481, Toronto, ON, M5T0S8
sex
male
tissue
brain, orbitofrontal cortex
disease
non-neurological disease control
extracted_molecule
genomic DNA
nuclei_isolation_procedure
Frozen orbitofrontal cortex (~50mg per sample) was dounce homogenized on ice in homogenization buffer (HB, 260mM sucrose, 30mM KCl, 10mM MgCl2, 20mM Tricine-KOH [pH 7.8], 1mM DTT, 0.5mM Spermidine, 0.15mM Spermine, 0.3% NP40, 1 tablet cOmplete Protease inhibitor [Sigma-Aldrich]) and then filtered using a 40m Flowmi cell strainer. Filtered nuclei were spun down at 500g for 5 min at 4C. Following removal of supernatant, nuclei were resuspended in 400L of HB and then mixed with 50% iodixanol (Sigma-Aldrich). The final nuclei suspension at 25% iodixanol was loaded onto a 30-40% iodixanol density gradient and then centrifuged at 3200g for 20 min at 4C. Nuclei collected from the 30-40% iodixanol interface were resuspended in HB and spun at 500g for 10 min at 4C. Supernatant was removed and the gradient purified nuclei were resuspended in diluted nuclei buffer (10X Genomics). Library preparation was performed by manufacturer instructions on the 10X Chromium Single Cell ATAC v1.1 platform. 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-atac mkfastq pipeline (10X Genomics). Reads were aligned to the GRCh38-2020-A genome and quantified using cellranger-atac count command on Cell Ranger ATAC v2.0 (10X Genomics).
data_processing
Fastq files were processed with CellRangerATAC 2.0.0 count function with GRCh38-2020-A-2.0.0 reference to produce the output files.
BioSampleModel
Human