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Sample GSM2046138

Query DataSets for GSM2046138

Status

Public on Jan 26, 2017

Title

U937-LT-2

Sample type

SRA

Source name

U937_LeTx treated

Organism

Homo sapiens

Characteristics

cell line: U937
cell type: human promonocytic lymphoma cell line
treated with: LeTx (1 μg/ml) for 24 hrs

Treatment protocol

Cells were treated with anthrax lethal toxin (1 μg/ml) for 24 hrs, and washed with RPMI medium to remove any residual toxins.

Growth protocol

Cells were seeded in 100 mm poly-L-lysine coated cell culture plate in RPMI medium containing 10% FBS (catalog # 26140), 100 IU/ml penicillin and 10 µg/ml streptomycin (catalog # 15140) from Invitrogen (CA, USA). The cells were maintained in a humidified incubator with a 95% air/5% CO2 atmosphere at 37°C. The medium was changed every 2-3 days. Cells were sub-plated and used for further experiments.

Extracted molecule

total RNA

Extraction protocol

RNA was extracted using RNAiso Plus (Takara, Shiga, Japan) and QIAGEN RNeasy® Mini kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. For RNA-Seq, RNA libraries were created from each group using the NEBNext® Ultra™ Directional RNA Library preparation kit from Illumina® (Illumina, San Diego, CA, USA). The first step in the workflow involved the removal of ribosomal RNA using the RNAMius™ Transcriptome Isolation kit (Life Technologies, Carlsbad, CA, USA). Following purification, total RNA was fragmented into small pieces using divalent cations at elevated temperature. The cleaved RNA fragments were copied into first-strand cDNA using reverse transcriptase and random primers, followed by second-strand cDNA synthesis using DNA polymerase I and RNase H. The cDNA fragments were then processed through an end-repair reaction by the addition of a single ‘A’ base, followed by ligation of the adapters. The products of these reactions were then purified and enriched by PCR to create the final cDNA library. The cDNA fragments were sequenced using the Illumina HiSeq2000 (101 cycles PE lane)
RNA libraries were prepared for sequencing using standard Illumina protocols

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

Illumina CASAVA v1.8.2 software was used for basecalling.
To avoid low-quality data, we clipped and trimmed the reads using Trimmomatic (v0.33)
For the analysis of differentially expressed genes, the data of quality-checked reads for each condition were processed with the TopHat (version 2.0.10) software based on reference genome sequence (Mus musculus UCSC mm10) and the differential gene expressed values of each sample were calculated by Cufflinks(v2.2.1)
the number of reads mapped to gene ID features were counted using HTSeq v0.6.1.
Genome_build: Homo sapiens UCSC hg19
Supplementary_files_format_and_content: tab-delimited text files including normalized read counts for each sample

Submission date

Jan 26, 2016

Last update date

May 15, 2019

Contact name

Se Kye Kim

E-mail(s)

skkim0217@gmail.com

Organization name

Hanyang University ERICA

Department

Molecular and Life Science