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Sample GSM8224492

Query DataSets for GSM8224492

Status

Public on Apr 26, 2024

Title

IGF113061

Sample type

SRA

Source name

Brain

Organism

Homo sapiens

Characteristics

tissue: Brain
braak: 5

Extracted molecule

nuclear RNA

Extraction protocol

Single nucleus isolation was conducted on 49 tissue blocks from (n=24, NDC; n=25, AD). Homologous fresh frozen brain tissue blocks from the EC, MTG and SSC were cryosectioned at 80um and 200mg of grey matter was collected in RNAse-free Eppendorf tube. All steps were carried out on ice or at 4°C. Tissue was homogenised in buffer (1% Triton X-100, 0.4 U/μl RNAseIn + 0.2 U/μl SUPERaseIn, 1ul 1mg/ml DAPI) using a 2ml glass douncer. The homogenate was centrifuged at 4°C for 8mins, 500g and supernatant removed. The pellet then was resuspended in homogenisation buffer and filtered through a 70um filter followed by density gradient centrifugation at 13,000g for 40mins. The supernatant was removed and nuclei were washed and filtered in PBS buffer (PBS + 0.5mg/ml BSA + 0.4 U/μl RNAseIn + 0.2 U/μl SUPERaseIn). Nuclei were pelleted, washed twice in PBS buffer and resuspended in 1ml PBS buffer. 100ul of nuclei solution was set aside on ice for single nuclear processing.
Isolated nuclei stained with Acridine Orange dye were counted on a LUNA-FL Dual Fluorescence Cell Counter (Logos Biosystems, L20001). Approximately 7000 nuclei were used for 10x Genomics Chromium Single Cell 3ʹ processing and library generation. All steps were conducted according to the 10x Genomics Chromium Single Cell 3' Reagent Kits v3 User Guide, with 8 cycles of cDNA amplification until fragmentation, where 25ng of amplified cDNA per sample was taken through for fragmentation. The final index PCR was conducted at 14 cycles. cDNA and library prep concentration were measured using Qubit dsDNA HS Assay Kit (ThermoFisher, Q32851) and DNA and library preparations were assessed using the Bioanalyzer High-Sensitivity DNA Kit (Agilent, 5067-4627). Pooled samples at equimolar concentrations were sequenced on an Illumina HiSeq 4000 according to the standard 10X Genomics protocol.
snRNA-seq

Library strategy

RNA-Seq

Library source

transcriptomic single cell

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

10X genomics

Data processing

Locally generated snRNASeq data were pre-processed using 10X Genomics Cell Ranger. Illumina sequencing files were aligned to the genomic sequence (introns and exons) using GRCh38 annotation in Cell Ranger v3.1. Nuclei were identified above background by the Cell Ranger software.
Assembly: GRCh38
Supplementary files format and content: count data and associated metadata

Submission date

Apr 23, 2024

Last update date

Apr 26, 2024

Contact name

Paul M Matthews

E-mail(s)

[email protected]

Organization name

Imperial College London

Street address

White City