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Sample GSM3415971

Query DataSets for GSM3415971

Status

Public on May 22, 2020

Title

DEK-rep2

Sample type

SRA

Source name

Skeletal muscle

Organism

Mus musculus

Characteristics

strain: Pax7CreERT2/+; H11DEK-EGFP/+
cell type: Muscle stem cells
treatment: PFA-perfused mice
age: 4.5-month-old

Extracted molecule

polyA RNA

Extraction protocol

The fixed muscle stem cell isolation was following the isolation protocol of satellite cells, as described previously in Liu et al., 2015, except that Collagenase II for first digestion was used at 2000 U/mL.Cell suspension was sorted using a BD Influx cell sorter equipped with 405-nm, 488-nm, 561-nm and 633-nm lasers. The machine was carefully optimized for purity and viability.
To get high quality RNA from fixed cells, the total RNA from fixed muscle stem cells were isolated using a miRNeasy FFPE Kit (Qiagen) following the protocol described in Thomsen et al., 2015. cDNA was generated following the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech) protocol. The RNA-Seq libraries were sheared with a Covaris S220 and generated using Ovation Ultralow DR Multiplex System 9-16 (NuGEN Technologies). The libraries were sequenced using Illumina NextSeq 500 (2 x 75).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

freshly-isolated quiescent muscle stem cells from PFA-perfused Pax7CreERT2/+; H11DEK-EGFP/+ mice

Data processing

RNA-seq data were aligned using STAR (version 2.5.3a) with the parameters “–twopassMode Basic”, “–outFilterIntronMotifs RemoveNoncanonical”, “--outSAMstrandField intronMotif”, and “--outSAMmultNmax 1”.
Then, StringTie (version 1.3.3) was used with the -e option to quantify gene expression levels according to the reference’s annotations.
Furthermore, StringTie’s -b option was used so that output suitable for Ballgown (version 2.10.0) under R (version 3.4.1) could be produced.
Genome_build: mm9
Supplementary_files_format_and_content: Table of genes by samples of FPKM values, as output by Ballgown. File format is tab-separated values (ASCII text file).

Submission date

Oct 03, 2018

Last update date

May 22, 2020

Contact name

Tom Cheung

Organization name

Hong Kong University of Science and Technology

Department

Division of Life Science

Street address

Clear Water Bay, Kowloon