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Sample GSM8377419

Query DataSets for GSM8377419

Status

Public on Sep 24, 2024

Title

muscle biopsy, overweight or obese male, before cold acclimation, subject 16

Sample type

SRA

Source name

Skeletal muscle biopsy

Organism

Homo sapiens

Characteristics

tissue: muscle
cell type: m. vastus lateralis biopsy
subjectid: Shiv16
moment of_sampling_(pre/post_intervention): Pre
Sex: Male
age (years): 67
bmi (kg/m2): 29.2
rna integrity_number_(rin): 7.6

Treatment protocol

Volunteers participated in a single-arm intervention study. During the study, participants were exposed for 10 days to an individualised cold exposure regimen that involved 1 hour of shivering per day via a cold-water (10°C) perfused suit. Before the intervention, and the day after 10 cold exposures with shivering (i.e. cold acclimation), a muscle biopsy was taken. Biopsies were taken following an overnight fast from the m. vastus lateralis, under local anaesthesia (1% lidocaine without adrenaline) using the Bergström method (PMID: 5584523). All samples were snap-frozen and stored at −80°C until the analyses were performed.

Growth protocol

In total, 15 overweight and obese males (n = 11) and females (n = 4) were included in the study. Prior to inclusion, all individuals were screened in an overnight fasted state to determine eligibility. Participants were white Western Europeans and aged between 40-75 years, with a BMI of 27-35 kg/m2. Females had to be postmenopausal, which was defined as at least one year post-cessation of menses. Exclusion criteria were: diagnosed with T2DM, cardiovascular disease (as determined by blood pressure measurements, resting ECG, and a medical history questionnaire), cancer, unstable body mass (weight gain or loss >3 kg in the past 3 months), fasting glucose concentration ≥7.0 mmol/L, anaemia, alcohol or drug abuse, engagement in structured exercise >2 hours per week, smoking, medication use regarded as unsafe in the context of the study and/or known to interfere with the primary outcome, and regular exposure to low temperatures such as cold-water baths, cold-water swimming or working in a refrigerated environment in the last one month. Participants started and completed the study between the 6th of November 2020 and the 13th of December 2021, at Maastricht University Medical Centre (Metabolic Research Unit), the Netherlands. The study was conducted in accordance with the declaration of Helsinki and was approved by the Medical Ethical Committee of Maastricht University Medical Centre. The study was registered at ClinicalTrials.gov (NCT-identifier: NCT04516018). All participants provided their written informed consent prior to the start of the screening.

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated using Trizol and further purified using Rneasy columns. Samples were only included for further analyses in case of intact bands corresponding to 18S and 28S ribosomal subunits and absence of chromosomal peaks or RNA degradation products.
Library construction and RNA sequencing on BGISEQ-500 were conducted at Beijing Genomics Institute (BGI) for pair-end 100bp runs. At BGI, Genomic DNA was removed with two digestions using Amplification grade DNAse I (Invitrogen). The RNA was sheared and reverse transcribed using random primers to obtain cDNA, which was used for library construction. The library quality was determined by using Bioanalyzer 2100 (Agilent Technologies). Then, the library was used for sequencing with the sequencing platform BGISEQ-500 (BGI).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

BGISEQ-500

Description

Skeletal muscle biopsy, obtained from the m. vastus lateralis, from an overweight male, before cold acclimation, subject 16
16v2

Data processing

Sequencing reads that contained adapters and/or had low quality, aligned to rRNA were filtered off before mapping.
The tool Salmon (version 1.9.0) was used to map the cleaned reads to the GRCh38.p13 human genome assembly-based transcriptome sequences as annotated by the GENCODE consortium (release 41). Salmon parameters used: -i ./salmon_index_H41 --libType A --seqBias --numBiasSamples 10000000 --gcBias --biasSpeedSamp 5 --validateMappings --numGibbsSamples 100
The Salmon-obtained transcript abundance estimates and lengths were imported in R using the package tximport to estimate gene level counts.
Assembly: GRCh38.p13
Supplementary files format and content: Matrix table (TxImport.GeneLevel.counts.GEO.txt) with raw gene counts for every gene and every sample (gene level counts were calculated from the Salmon-obtained transcript abundance estimates)
Supplementary files format and content: R-object (txi.counts.annotated.Rdata; exported tximport object) containing gene level counts estimated from the Salmon-obtained transcript abundance estimates. Note: count data was not transformed nor scaled (i.e. countsFromAbundance = "no"), and can be used for subsequent analysis in edgeR or DESeq2.
Supplementary files format and content: R object (txi.lengthScaledTPM.annotated.Rdata; exported tximport object) containing scaled gene level counts estimated from the Salmon-obtained transcript abundance estimates. Note: count data was scaled using library size and transcript gene length (i.e. countsFromAbundance = "lengthScaledTPM"), and these scaled counts can be used for subsequent analysis in limma (voom function).

Submission date

Jul 03, 2024

Last update date

Sep 24, 2024

Contact name

Guido Hooiveld

E-mail(s)

[email protected]

Organization name

Wageningen University

Department

Div. Human Nutrition & Health