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Sample GSM4585062

Query DataSets for GSM4585062

Status

Public on Jun 02, 2020

Title

hiPSCs_ctrl_rep1

Sample type

SRA

Source name

585A1 hiPSCs

Organism

Homo sapiens

Characteristics

conditions: self-renewal condition
treatment: none

Treatment protocol

0.5 MV/cm of THz light was irradiated to hiPSCs.

Growth protocol

585A1 hiPSCs were maintained in mTeSR-1-defined medium (Stem Cell Technologies).

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted from differentiated primary myoblasts using the RNeasy mini kit (QIAGEN).
To prepare the cDNA library, 10 ng of total RNA was diluted with 9 µL of RNase free water, then mixed with VN primer (Oxford NANOPORE Technologies, UK), 1 µL of 10 mM dNTPs (New England Biolabs Inc. Ipswich, Massachusetts, USA), and incubated at 65˚C for 5 min as the RNA solution. Separately, 4 µL of 5x RT Buffer (Thermo Fisher Scientific), 1 µL of RNaseOUT (Thermo Fisher Scientific), 1 µL of Nuclease-free water and 2 µL of Strand-Switching Primer (Oxford NANOPORE Technologies) was mixed as the strand-switching buffer. The two solutions were mixed at 42˚C for 2 min; then, 1 µL of Maxima H Minus Reverse Transcriptase (Thermo Fisher Scientific) was added, incubated at 42˚C for 90 min, 85˚C for 5 min, and stored at 4˚C until use as the cDNA library.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

MinION

Description

none_rep1
RNA obtained from 585A1 hiPSCs under mTeSR-1 self-renewal condition, replicate 1

Data processing

Base-calling and demultiplexing were performed with EPI2ME software (Oxford NANOPORE Technologies).
Failed reads were discarded and Fast5 files were converted into FASTQ and FASTA files using EPI2ME software.
Generated FASTQ files were loaded to BioJupies for alignment with human genomes and annotation.
the counts were analysed using TCC in R Bioconducter. Briefly, the counted data set was introduced into the TCC-GUI package.
Genome_build: human_matrix.h5 v2
Supplementary_files_format_and_content: Gene counts were normalized using TMM (Trimmed mean of M values) methods in the edgeR package with the parameters: Number of Iterations = 3; FDR < 0.1; Elimination of Potential DEGs = 0.05.

Submission date

Jun 01, 2020

Last update date

Jun 02, 2020

Contact name

Ken-ichiro Kamei

E-mail(s)

kamei.kenichiro.7r@kyoto-u.ac.jp

Phone

+81-757539774

Organization name

Kyoto University