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Sample GSM5403839

Query DataSets for GSM5403839

Status

Public on Aug 10, 2021

Title

primary skeletal myotubes, bOHB treatment, replicate 3

Sample type

SRA

Source name

primary mouse skeletal myotubes, treated with β-hydroxybutyric acid (bOHB; 5mM) for 6 hrs

Organism

Mus musculus

Characteristics

strain: C57Bl/6
cell type: primary skeletal myotubes
treatment: 5 mM bOHB [(R)-(–)-3-Hydroxybutyric acid sodium salt]
exposure time: 6 hrs

Treatment protocol

Primary cells were treated for 6h with 5 mM βOHB or Butyrate, with PBS as control. Adipocytes and Macrophages were treated in DMEM/FCS/PS. Myotubes were treated in DM. Hepatocytes were treated in Williams E medium. Cells were washed with PBS once and stored in -80 °C until RNA was isolated.

Growth protocol

Primary mouse adipocytes (isolation and differentiation of stromal vascular fraction): Inguinal white adipose tissue from 3-4 C57Bl/6 male mice was collected and placed in Dulbecco’s modified eagle’s medium (DMEM; Lonza) supplemented with 1% Penicillin/Streptomycin (PS) and 1% bovine serum albumin (BSA; Sigma-Aldrich). Material was minced finely with scissors and digested in collagenase-containing medium (DMEM with 3.2 mM CaCl2, 1.5 mg/ml collagenase type II (C6885, Sigma-Aldrich), 10% FBS, 0.5% BSA, and 15 mM HEPES) for 1 h at 37°C, with occasional vortexing. Cells were filtered through a 100-μm cell strainer (Falcon). Subsequently, the cell suspension was centrifuged at 1600 rpm for 10 min and the pellet was resuspended in erythrocyte lysis buffer (155 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA). Upon incubation for 2 min at room temperature, cells were centrifuged at 1200 rpm for 5 min and the pelleted cells were resuspended in DMEM containing 10% fetal bovine serum (FBS) and 1% PS (DMEM/FBS/PS) and plated. Upon confluence, the cells were differentiated according to the protocol as described previously [PMID: 22569073 and 23150577]. Briefly, confluent SVFs were plated in 1:1 surface ratio, and differentiation was induced 2 days afterwards by switching to a differentiation induction cocktail (DMEM/FBS/PS, 0.5 mM isobutylmethylxanthine, 1 μM dexamethasone, 7 μg/ml insulin and 1 µM rosiglitazone) for 3 days. Subsequently, cells were maintained in DMEM/FBS/PS, and 7 μg/ml insulin for 3-6 days and switched to DMEM/FBS/PS for 3 days. Average rate of differentiation was at least 80% as determined by eye. Primary mouse macrophages (isolation and differentiation of bone marrow derived monocytes): Bone marrow cells were isolated from femurs of C57Bl/6 male mice following standard protocol and differentiated into macrophages (bone marrow-derived macrophages, BMDMs) in 6-8 days in DMEM/FBS/PS supplemented with 20% L929-conditioned medium (L929). After 6-8 days, non-adherent cells were removed, and adherent cells were washed and plated in 12-well plates in DMEM/FBS/PS + 10% L929. After 24 hours, medium was switched to 2% L929 in DMEM/FBS/PS overnight. Cells were treated the following day. Primary skeletal myotubes (isolation and differentiation of skeletal myocytes): Myoblasts from hindlimb muscle of WT-C57Bl/6 male mice were isolated as previously described [PMID: 29879622]. In brief, the muscles were excised, washed in 1× PBS, minced thoroughly, and digested using 1.5 mL collagenase digestion buffer (500 U/ml or 4 mg/mL collagenase type II (C6885, Sigma-Aldrich), 1.5 U/ml or 5 mg/mL Dispase II (D4693, Sigma-Aldrich), and 2.5 mM CaCl2 in 1× PBS) at 37°C water bath for 1 h in a 50 ml tube, agitating the tube every 5 min. After digestion, the cell suspension containing small pieces of muscle tissue was diluted in proliferation medium (PM: Ham's F-10 Nutrient Mix (#31550023, Thermo Fisher Scientific) supplemented with 20% fetal calf serum, 10% HS, 0.5% sterile filtered chicken embryo extract (#092850145, MP Biomedicals), 2.5 ng/ml basic fibroblast growth factor (#PHG0367, Thermo Fisher Scientific), 1% gentamycin, and 1% PS), and the suspension was seeded onto matrigel-coated (0.9 mg/ml, #354234, Corning) T150 flasks at 20% surface coverage. Cells were grown in 5% CO2 incubator at 37°C. Confluence was reached latest after 5 d in culture, upon which cells were trypsinized (0.25% trypsin), filtered with 70 µm filters, centrifuged at 300 g for 5 min, and then seeded on an uncoated T150 flask for 45 min to get rid of fibroblasts. Subsequently, myoblasts were seeded in PM at 150.000 cells/mL onto matrigel-coated 12-well plates cells. Upon reaching confluence, differentiation was induced by switching to differentiation medium (DM: Ham's F-10 Nutrient Mix supplemented with 5% horse serum (HS) and 1% PS). DM was replaced every other day. Myotubes fully differentiated by Day 5 of differentiation in DM. The medium was renewed every other day. Primary mouse hepatocytes (isolation and culturing of hepatocytes): Primary hepatocytes were isolated from C57BL/6NHsd male mice via collagenase perfusion. Cells were plated onto collagen (0.9 mg/ml) coated 24-well plates at 200,000 cells/well in Williams E medium (PAN Biotech), substituted with 10% FBS, 100 nM dexamethasone and penicillin/streptomycin and maintained at 37 °C in an atmosphere with 5% CO2. After four hours of attachment, cells were washed with phosphate buffer saline (PBS) and allowed to rest in dexamethasone-free medium overnight before treatment.

Extracted molecule

total RNA

Extraction protocol

Total RNA from all cell culture samples was extracted using TRIzol reagent (Thermo Fisher Scientific) and purified using the Qiagen RNeasy Mini kit (Qiagen) according to manufacturer’s instructions. RNA concentration was measured with a Nanodrop 1000 spectrometer and RNA integrity was determined using an Agilent 2100 Bioanalyzer with RNA 6000 microchips (Agilent Technologies).
Library construction and RNA sequencing on BGISEQ-500 were conducted at Beijing Genomics Institute (BGI) for pair-end 150bp 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

Data processing

Sequencing reads that contained adapters and/or had low quality, aligned to rRNA were filtered off before mapping.
The tool Salmon (version 1.2.1) was used to map the cleaned reads to the GRCm38.p6 mouse genome assembly-based transcriptome sequences as annotated by the GENCODE consortium (release M25). Salmon parameters used: -i ./salmon_index_M25 --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 (version 1.16.1) to estimate gene level counts.
Genome_build: GRCm38.p6
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample (gene level counts were calculated from the Salmon-obtained transcript abundance estimates; TxImport.GeneLevel.counts.GEO.txt)
Supplementary_files_format_and_content: R-object (txi.counts.annotated.GEO.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.GEO.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

Jun 28, 2021

Last update date

Aug 10, 2021

Contact name

Guido Hooiveld

E-mail(s)

guido.hooiveld@wur.nl

Organization name

Wageningen University

Department

Div. Human Nutrition & Health