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Sample GSM3978699

Query DataSets for GSM3978699

Status

Public on May 17, 2021

Title

29_OB6CNT_Parabiosis_RNASeq [29_S44]

Sample type

SRA

Source name

Old B6 control

Organism

Mus musculus

Characteristics

age at procedure: 22 months old
parabiont status: Old B6 control
Sex: Male
cell type: Hematopoietic stem cells

Extracted molecule

total RNA

Extraction protocol

Hematopoietic stem cells were isolated from the bone marrow of mice through a standardized process of bone marrow extraction, creation of a single cell suspension, and flow cytometry based cell sorting. RNA was isolated using the PicoPure RNA Isolation Kit from ThermoFisher.
Purified RNA was used as input for the Nugen Ovation Trio low input RNA library preparation kit. Samples were processed on an automated robot system at the NYU Genome Technology Center

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

29_S44_Quants

Data processing

Fastq files were ran through fastqc to check for sequencing quality. 5 of 35 samples (numbers 6, 15, 16, 27, 35) were omitted due to poor quality and / or low coverage. QC was visualized using MultiQC (Ewels et al Bioinformatics 2016)
Salmon (Patro et al Nature Methods 2017) was used to generate an index of the CHR Gencode mouse transcriptome (gencode.vM20.trasncripts.fa)
Salmon was used to quanitfy transcripts against the mouse transcriptome reference. Flags used were for automatic library type detection -l A (unstranded, single-end), --seqBias, --gcBias, and --validateMappings
Transcript quantification files (quants.sf) were convereted to gene level counts using TxImport (Soneson et al. F1000Research 2015)
Differential expresison on gene level counts was performed using DESeq2 (Love et al Genome Biology 2014). Results were generated using the lfcShrink and contrast functions, making pairwise comparsions on the indicated groups. Alpha threshold set to 0.05 and lfcThreshold to 1.
Genome_build: Read alignment was not utlized for this analysis. Gencode utilizes the GRCm38 primary assembly genome sequence as the reference for their transcriptome annotation.
Supplementary_files_format_and_content: Differential expression files include log2 fold changes generated by DESeq2 for each indicated comparison (with all samples from a given group identified by parabiont status utilized in the differential expression test). These files are listed as .csv files. A counts file is also provided with all the gene level counts for each sample that are used as the input for DESeq2, also as a .csv file.

Submission date

Jul 25, 2019

Last update date

May 17, 2021

Contact name

Paul Vincent Dellorusso

E-mail(s)

[email protected]

Phone

5166552491

Organization name

Columbia University Irving Medical Center