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Sample GSM6507020

Query DataSets for GSM6507020

Status

Public on Aug 27, 2022

Title

NAcc sample from mouse 1 [S1_NAcc]

Sample type

SRA

Source name

Brain

Organism

Mus musculus

Characteristics

tissue: Brain
region: NAcc
genotype: B6
Sex: female
treatment: naïve
sequencing tray: 2
animal id: 1

Extracted molecule

polyA RNA

Extraction protocol

RNA were extracted from each brain region using the QIASymphony robot and standard Qiagen kits/reagents (Qiagen Sciences Inc, Germantown, MD) following the manufacturer’s recommendations. Brain tissue were obtained from 1-mm coronal slices by hand under RNAse-free conditions. (Gene Profiling Shared Resource, Oregon Health & Science University)
RNA libraries were prepared with a TruSeq stranded mRNA library prep Kit (cat# RS-122-2101, Illumina, San Diego, CA, USA).
RNA-Seq: The libraries were sequenced on a Illumina NovaSeq 6000 (Massively Parallel Sequencing Shared Resource, Oregon Health & Science University) using a paried-end read.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

HSCC_Founders_STAR_Counts.txt

Data processing

Sequences raw files were imported into PARC's Prime‐Seq, LabKey Server‐based system (Nelson EK, Piehler B, Eckels J, et al. LabKey server: an open source platform for scientific data integration, analysis and collaboration. BMC Bioinformatics. 2011;12(1):71.)
Quality control of the sequences was verified through FastQC (Andrews, 2010). The mean counts per sample in sequencing tray 3 was found to be low. Trays 3 and 2 (as a common reference) were resequenced. Original tray 3 and rerun tray 2 were excluded from further analysis.
Illumina adapters were removed and 3' read ends were trimmed using Trimmomatic (Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30(15):2114‐2120)
Reads were aligned to the Mus Musculus reference genome (Ensemble_Mouse_GRCm39_GCA_000001635.9) using the STAR aligner (v. 2.7.3a) allowing for maximum of 3 mismatches and only unique alignments.
Per‐sample gene count tables were generated by STAR using Ensembl gene annotations (Mus_musculus.GRCm39.105) and were merged to form a single gene count matrix.
Genes with either: (1) less than 1 count-per-million across samples, or (2) above 50,000 counts in a single sample were removed from further analysis.
One sample (S1_NAcc) with an inter-array correlation greater than 3 standard deviations from the mean was excluded from futher analysis as outliers.
Trimmed mean of M-values and voom normalization was performed using the edgeR (v. 3.38.1) Bioconductor package.
Differential expression (DE) between all possible 28 pairwise combiantions of the eight HS-CC founder strains was determined using the limma+voom pipeline (v. 3.52.2). Log fold change and other statistics was computed using topTable or topTreat as appropriate. Top level comparison was done using decideTests using the "global" method. The Benjamini-Hochberg method was used to determine the false discovery rate.
Assembly: Mmul10
Supplementary files format and content: HSCC_Founders_STAR_Counts.txt, STAR alignment counts for all 240 samples (columns) for all 55414 genes (rows).
Supplementary files format and content: HSCC_Founders_Normalized_Counts.txt, Normalized(TMM+voom) counts for all 143 samples (columns) and 14785 genes (rows) used in differential expression analysis.

Submission date

Aug 24, 2022

Last update date

Aug 27, 2022

Contact name

Justin Anderson

E-mail(s)

andejsut@ohsu.edu

Organization name

Oregon Health & Science University

Department

Behavorial Neuroscience