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Sample GSM5140571

Query DataSets for GSM5140571

Status

Public on Jun 20, 2022

Title

Control_lot1

Sample type

SRA

Source name

C2C12 cell line

Organism

Mus musculus

Characteristics

cell line: C2C12
cell type: myoblast

Treatment protocol

For time series analysis, cells were washed twice with PBS and treated with 200 µL of 0.25% trypsin for 30 sec, 3 min or 30 min at 37ºC.
For low trypsin level treatment, cells were washed twice with PBS, and treated with 200 µL of 0.25% trypsin. Trypsin was removed immediately and cells were incubated at 37 °C for 3 min.
For cold-active protease treatment, cells were washed twice with PBS followed by adding 200 µL of cold protease. Cells were incubated at 4 °C for 5 min with shaking.

Growth protocol

C2C12 cells were cultured in 6 well plates in Dulbecco’s modified Eagle’s medium (DMEM) (1g/L D-glucose) supplemented with 20% fetal bovine serum (FBS), 2 mM L-glutamine, and 1% penicillin-streptomycin.

Extracted molecule

total RNA

Extraction protocol

All samples were collected by adding lysis buffer to the cell culture plates followed by scraping (Control) and re-suspending cells. Total RNAs were extracted from lysate using NucleoSpin RNA XS (Takara).
Sequencing was performed using HiSeq PE Rapid Cluster Kit v2 and HiSeq Rapid SBS Kit v2 (50 Cycles) on Illumina HiSeq 1500 sequencers, with 15 cycles for read 1 and 45 cycles for read 2.

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina HiSeq 1500

Data processing

Library strategy: BRB-seq
Extraction of sample barcodes and unique-molecular-identifiers (UMIs) was performed using UMI-tools (version 1.0.1) with the following commands: umi_tools extract -I r1.fastq --read2-in=r2.fastq --bc-pattern=NNNNNNCCCCCC --read2-stdout.
The reads were trimmed using Cutadapt (M. Martin, 2011) (version 2.6) wapper, Trim Galore! (version 0.6.6), with the option: -a GATCGTCGGACT. The reads were mapped by the aligning software HISAT2 (D. Kim et al., 2015, M. Pertea et al., 2016, D. Kim et al., 2019) (version 2.1.0) to the reference genome (GRCm38, pre-built HISAT2 index, genome_snp_tran).
Read counts per gene were obtained using featureCounts (Liao Y et al., 2014) (version 2.0.1). UMIs were not used and mitochondrial genes are excluded in the following analysis.
Downstream analysis was conducted by DESeq2 software (M. I. Love et al., 2014) (1.28.1).
Genome_build: GRCm38/mm10
Supplementary_files_format_and_content: *.csv file include comma-delimited RNA-seq read count matrix, normalized count matrix, and TPM matrix.

Submission date

Mar 08, 2021

Last update date

Jun 20, 2022

Contact name

Yasuyuki Ohkawa

E-mail(s)

yohkawa@bioreg.kyushu-u.ac.jp

Organization name

Medical Institute of Bioregulation

Lab

Division of Transcriptomics