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Sample GSM7840044

Query DataSets for GSM7840044

Status

Public on Oct 17, 2023

Title

HEK293 cells, wild type, basal, biol rep 2

Sample type

SRA

Source name

wild type

Organism

Homo sapiens

Characteristics

cell line: HEK293
cell type: embryonic
genotype: wild type
treatment: incubation with vehicle
time: 1 h

Treatment protocol

Cells were seeded in poly-D-lysine coated 6 well-plates and transfected with wild type β2AR plasmid. The following day, the cells were serum starved overnight. After 48 hours of transfection, the cells were incubated with 1 μM isoproterenol or vehicle for 1 h.

Growth protocol

HEK293 wild type, β-arrestin1/2 KO and Gαs KO cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (D6429, Sigma) supplemented with 10% fetal bovine serum (FBS) (F2442, Sigma) and 1% penicillin/streptomycin (A5955, Sigma) and maintained in humidified atmosphere with 5% CO2 at 37°C.

Extracted molecule

total RNA

Extraction protocol

After stimulus, the cells were washed with PBS, total RNA was isolated with a RNeasy Mini Kit (74104, Qiagen) including an on-column DNase I digestion and quantified using a Nanodrop ND-1000 (Thermo Scientific).
mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries were pooled and sequenced on Illumina Novaseq 6000 platform, according to effective library concentration and data amount.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

Wild type cells basal 2

Data processing

The quantification of transcripts was calculated using Salmon (version 1.7.0), which provides accurate expression estimates.
The differential gene expression analysis including quality control, model fitting, and hypothesis testing was conducted using DESeq2.
Assembly: Homo sapiens (human) genome assembly GRCh38 (hg38) from Genome Reference Consortium
Supplementary files format and content: CSV file containing merged count values from all quant.sf files generated by Salmon. NOTE: Aligned against spliced transcripts

Submission date

Oct 12, 2023

Last update date

Oct 17, 2023

Contact name

Valeria Burghi

E-mail(s)

valeburghi@gmail.com

Organization name

University of California San Diego

Department

Pharmacology