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Sample GSM5176983

Query DataSets for GSM5176983

Status

Public on Mar 19, 2021

Title

NC ctrl 72h 3

Sample type

SRA

Source name

primary brite adipocytes

Organism

Mus musculus

Characteristics

source organ: inguinal WAT
genotype: 129SvEv

Treatment protocol

Cells were reverse transfected with lncRNA Ctcflos-targeting or non-targeting control LNA Gapmer Antisense oligonucleotides [50nM] and harvested either 24 hours or 72 hours post transfection.

Growth protocol

Primary preadipocytes were prepared from stromal-vascular fraction of murine inguinal white adipose tissue of 129SvEv mice, induced at sub-confluence for 2 days and differentiated for 1 day in the presence of rosiglitazone to induce browning.

Extracted molecule

total RNA

Extraction protocol

Differentiating brite adipocytes were washed with PBS and scraped into Trisure (Bioline). Total RNA was prepared from Trisure (Bioline) and column-purified (SV Total RNA Isolation System, Promega).
Libraries were prepared from total RNA by tagmentation of full-length ds cDNA (5 ng) with Tn5 enzyme (1 µl, 11 µM) and amplified (15 cycles). AMPure beads (Beckman Coulter, #A63881) were used to size-select fragments of 200-1000 bp in two rounds (0.5x beads and 0.7x beads, respectively). Libraries were profiled with the High Sensitivity NGS Fragment Analysis Kit (Advanced Analytical, #DNF-474), measured with the Qubit dsDNA HS Assay Kit (Invitrogen, #Q32851), pooled and sequenced on the Illumina NextSeq 500 platform with custom primer and High Output v2 kit (75 cycles) (Illumina, #FC-404-2005).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

Sequencing on the Illumina NextSeq 500 platform with custom primer and High Output v2 kit (75 cycles) (Illumina, #FC-404-2005), Reads from barcoded mRNA-seq experiments have two barcodes, corresponding to the two levels of multiplexing. The first one is common to standard protocols and is used to separate the libraries. The second is specific to the barcoded mRNA-seq protocol and is used to separate the multiplexed samples from the bulk data.
The first demultiplexing step was performed with the Illumina BaseSpace platform, while the second was performed using custom scripts. FastQ files containing 62 bp long reads were analysed.
Sequenced reads were mapped to the murine genome, NCBI build 38, by the Genomatix Mapper 3.7.6 (Genomatix Mining Station, settings: 'deep', min. quality 92%).
Unique hits were subjected to expression analysis for RNA-seq (Genomatix Genome Analyzer, ElDorado version 12-2013) and provided as reads per kilobase of exon per megabase of library size (RPKM).
Genome_build: murine NCBI genome build 38
Supplementary_files_format_and_content: tab-delimited text file providing RPKM values for each sample.

Submission date

Mar 18, 2021

Last update date

Mar 19, 2021

Contact name

Tobias Fromme

E-mail(s)

[email protected]

Organization name

Technical University of Munich

Department

Molecular Nutritional Medicine