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Sample GSM5972670

Query DataSets for GSM5972670

Status

Public on Jan 19, 2023

Title

15Run_e

Sample type

SRA

Source name

Murine inguinal white adipose tissue from exercised animals sampled at ZT15

Organism

Mus musculus

Characteristics

tissue: inguinal white adipose tissue
Sex: male
strain: C57BL6
sampling time: ZT15
exercise: RUN

Extracted molecule

total RNA

Extraction protocol

Inguinal adipose tissue was used for RNA extraction. Tissues were homogenized using TissueLyser (Qiagen) in 1 mL TRIzol (Sigma-Aldrich). Chloroform was added, samples were mixed, subjected to centrifugation, and the aqueous phase was transferred to a new tube. Thereafter, 1:1 70% ethanol was added, and the total volume was loaded on silica-membrane columns. RNA extraction was performed using a commercially available kit (Rneasy lipid tissue kit, Qiagen). DNase treatment was performed. RNA concentration and purity were assessed by absorbance at 260 and 280 nm using NanoDrop One (Thermo Fisher Scientific, Waltham, MA).
Aliquots of RNA (1000 ng) were analyzed using the Illumina TruSeq Stranded Total RNA with Ribo-Zero Gold protocol (Illumina) as described (ref). Ribosomal RNA was removed from the sample using 35 μl of rRNA removal beads (Illumina) on a magnetic plate followed by cleanup of the ribosomal-depleted RNA with 193 μl of Agencourt RNAClean XP beads (Beckman Coulter), 70% ethanol wash, and elution into 10 μl elution buffer (Illumina). The RNA sample was fragmented for 4 min at 94°C in Elute, Prime, Fragment High Mix (Illumina) and then subjected to first-strand cDNA synthesis with 1 μl of Superscript III reverse transcriptase (Life Technologies) per sample using a thermocycler programmed to 25°C for 10 min, 50°C for 15 min, and 70°C for 15 min. Second-strand cDNA was synthesized by addition of Second-Strand Marking Master Mix, and samples were incubated at 16°C for 60 min. Samples were subjected to another bead cleanup before A-tailing and ligation of adapters as per kit instructions (Illumina). Following a third bead cleanup, samples were enriched for DNA fragments by amplification using the Illumina polymerase chain reaction (PCR) Primer Cocktail and PCR Master Mix, subjected to 98°C for 30 min, followed by a predefined cycle (98°C for 10 s, 60°C for 30 s, and 72°C for 30 s) that was repeated 3 to 15 times, on the basis of each individual sample, and finally incubated for 5 min at 72°C. Samples were cleaned and validated for DNA concentration using the Qubit dsDNA HS assay kit (Invitrogen) and for base pair size and purity using the Agilent High Sensitivity DNA chip and Bioanalyzer instrument.
38-bp paired-end sequencing on a NextSeq500

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

star/2.7.2b STAR --outReadsUnmapped Fastx --outSAMstrandField intronMotif--outFilterIntronMotifs RemoveNoncanonicalUnannotated --outSAMtype BAM SortedByCoordinate
subread/1.6.2 featureCounts -p -B -C -T $threads -t exon -g gene_id
limma_3.48.3 filterByExpr.DGEList(dataDGE, design = model.matrix(~0 + group + sex), min.count = 15)
Assembly: mm 10, release 99
Supplementary files format and content: processed.data.csv contains cpm after filtering by expression with cutoff at 10 counts along with HUGO symbol, ensmbl, and entrezID

Submission date

Mar 25, 2022

Last update date

Jan 19, 2023

Contact name

Leonidas S Lundell

E-mail(s)

leonidas.lundell@gmail.com

Organization name

Karolinska Institute

Department

Physiology and Pharmacology