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Sample GSM4123934

Query DataSets for GSM4123934

Status

Public on Jun 01, 2020

Title

FOXP2 dhelix + CHIR rep3

Sample type

SRA

Source name

U2OS

Organism

Homo sapiens

Characteristics

cell type: osteosarcoma epithelial cells
cell line: U2OS
genotype: FOXP2 dhelix (lacking residue 264-272) overexpression + CHIR treatment

Treatment protocol

In a six-well plate, approx. 80% confluent U2OS cells were transfected with various FOXP2 construct plasmids (500 ng) in equal amount using Lipofectamin 3000 (Thermo Fisher Scientific) for 48h. In case of CHIR-treatment, 5µM CHIR 99021 was added in the medium 72h before transfection with FOXP2 construct plasmids.

Growth protocol

Human Osteosarcoma Epithelial cells (U2OS) were maintained at 37°C under 5% CO2 in α-MEM medium (Invitrogen) supplemented with 5% (v/v) FCS (Gibco), 100 IU/ml penicillin and 100 µg/ml streptomycin.

Extracted molecule

total RNA

Extraction protocol

QIAshredder and RNeasy Mini Kit (Qiagen, UK) was used to isolate RNA from transfected U2OS cells according to the manufacturer’s protocol. Integrity of RNA was determined using a bioanalyzer instrument (Agilent Technologies, Santa Clara, CA, USA).
A cDNA library including 5 replicates per condition was prepared using the TruSeq Stranded mRNA Sample Prep Kit (Illumina Inc., San Diego, CA) according to the manufacturer´s recommendation. Briefly, 1µg of total RNA was used for first-strand synthesis performed with a random hexamer and SuperScript II (Life Technologies, Carlsbad, CA, USA). Second-strand synthesis was performed using dUTP and the Illumina-specific Second Strand Marking Master Mix. After end repair and A-tailing indexed adaptors were ligated to the cDNA fragments. Fragments successfully ligated with adaptor molecules on both ends were enriched by PCR for 15 cycles and purified with AMPure XP Beads (Beckman Coulter Inc., Brea, CA). The final libraries were quality checked on an Agilent Bioanalyzer and quantified with qPCR using a commercially available PhiX-library (Illumina Inc., San Diego, CA) as a reference. All samples were run in five biological replicates and a total of 30 equimolarily pooled samples were sequenced two High Output flow cells on an Illumina NextSeq in a paired end run with 2x 75 cycles.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

NextSeq 550

Data processing

Alignment: Star 2.6.1a_08-27
Transcript Counts: featureCounts (subread package) 1.4.4
Raw RNA-Seq reads were aligned to the human hg19 genome using STAR. considered differentially expressed.
Gene read counts are generated using featureCounts and differential expression was analyzed using DESeq2.
Genes with adjusted p-value lower than 0.05 and a log2 fold change of 1 or -1 were considered differentially expressed.
Genome_build: hg19
Supplementary_files_format_and_content: read counts per gene

Submission date

Oct 16, 2019

Last update date

Jun 01, 2020

Contact name

Gesa Richter

E-mail(s)

gesa.richter@medunigraz.at

Organization name

Medical University Graz

Street address

Neue Stiftingtalstrasse 6