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Sample GSM8120860

Query DataSets for GSM8120860

Status

Public on Apr 19, 2024

Title

KO, Saline, 12mo, rep 3

Sample type

SRA

Source name

cortex

Organism

Mus musculus

Characteristics

tissue: cortex
genotype: Grn KO
treatment: Saline
treatment dose: None
rin: 9.3
age: 12.67 months

Treatment protocol

AAV was applied intravenously via tail vain injection (max. 200 µl). For tissue collection, mice were anesthetized with either Ketamine/Xylazine or tribromoethanol. Mice were transcardiacally perfused with chilled PBS before organ collection. Tissues and fluids were stored at -80°C until further use.

Growth protocol

All animal experiments were performed either according to German animal welfare law and approved by the government of upper Bavaria (Vet_02-17-106) or by the Denali Therapeutics Institutional Animal Care and Use Committee. Mice were kept under standard housing conditions; standard pellet food and water was provided ad libitum.

Extracted molecule

polyA RNA

Extraction protocol

Total RNA was extracted from frozen brain powder using the Qiagen RNeasy plus kit (Qiagen, 74034). Quality and quantity were assessed with a 4200 TapeStation System (Agilent).
Next generation sequencing libraries were generated with the QuantSeq 3' mRNA-seq Library Prep Kit FWD for Illumina (Lexogen A01173), with the UMI second strand synthesis module in order to identify and remove PCR duplicates. Library quantity and quality were assessed with the 4200 TapeStation system (Agilent). Libraries were pooled in equimolar ratios for sequencing.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

DRN-17105

Data processing

Sequencing adapters were trimmed from the raw reads with skewer (version 0.2.2).
Unique molecular identifiers (UMIs) were extracted from each read with the umi2index tool (Lexogen). Quality control of the trimmed reads was performed using FastQC (version 0.11.5).
Reads were aligned to the mouse genome version GRCm38_p6. A STAR index (version 2.7.1a) was built with the –sjdbOverhang=50 argument. Splice junctions from Gencode gene models (release M17) were provided via the –sjdbGTFfile argument. STAR alignments were generated with the following parameters: –outFilterType BySJout, –quantMode TranscriptomeSAM, –outFilterIntronMotifs RemoveNoncanonicalUnannotated, –outSAMstrandField intronMotif, –outSAMattributes NH HI AS nM MD XS and –outSAMunmapped Within. Alignments were obtained with the following parameters: –readFilesCommand zcat –outFilterType BySJout –outFilterMultimapNmax 20 –alignSJoverhangMin 8 –alignSJDBoverhangMin 1 –outFilterMismatchNmax 999 –outFilterMismatchNoverLmax 0.6 –alignIntronMin 20 –alignIntronMax 1000000 –alignMatesGapMax 1000000 –quantMode GeneCounts –outSAMunmapped Within –outSAMattributes NH HI AS nM MD XS –outSAMstrandField intronMotif –outSAMtype BAM SortedByCoordinate –outBAMcompression 6.
Alignments sharing the same UMI and genomic coordinate were deduplicated using the collapse_UMI_bam tool (Lexogen). Post-alignment quality control reports were generated using MultiQC (version 1.0).
The raw gene expression matrix was constructed from the 'forward' column of STAR's ReadsPerGene.out.tab output files using R (version 4.2). Gene symbols and biotype information were extracted from the Gencode GTF file. To calculate normalized counts per million (CPM) TMM normalization was applied across all genes annotated as protein_coding with the edgeR R package (version 3.40.2).
Assembly: GRCm38
Supplementary files format and content: Microsoft excel format with three worksheets: 1) sample annotations, 2) raw counts, 3) normalized counts (CPMs)

Submission date

Feb 29, 2024

Last update date

Apr 19, 2024

Contact name

Thomas Sandmann

E-mail(s)

genomics@dnli.com, sandmann@dnli.com

Organization name

Denali Therapeutics

Street address

161 Oyster Point Blvd