cell type: myotube lot number: Lonza lot #20TL182717
Treatment protocol
After five days of differentiation, various concentrations of ITLN1 (i.e., 0, 10, 100 and 500ng/ml) were added to the skeletal muscle cells for six hours.
Growth protocol
Primary human skeletal muscle cells (SkMCs) sourced from three different healthy donors (lot 20TL182717, 20TL070666 and 18TL180368) were purchased from Lonza (CC-2580, UK) and cultured as per the manufacturer’s protocol. Briefly, the cells were incubated at 37ᵒC and 5% CO2 in growth medium (CC-3245, Lonza, UK) and passaged when a confluence of 80% was reached. The cells were washed with Dulbecco's phosphate-buffered saline (DPBS, 14040117, Thermo Fisher, UK) and the culture medium was replaced every 48 hours. After four passages, the myoblasts were harvested using Gibco’s trypsin-EDTA (25200056, Thermo Fisher, UK), counted with a Bio-Rad TC20 automated cell counter using cell counting slides (Bio-Rad, 1450016, UK) and transferred to 6-well or 96-well (polystyrene) plates. To induce differentiation, the cells were incubated in Dulbecco's Modified Eagle Medium/Ham's F-12 supplemented with 2% horse serum (DMEM, 12634010, Thermo Fisher, UK) for 3 to 5 days depending on protocol requirements.
Extracted molecule
total RNA
Extraction protocol
Following incubation with intelectin treatment, the RNeasy mini kit (Qiagen, 173046459, UK) was used to extract and purify total RNA. Briefly, the myotubes were lysed and homogenised with a highly denaturing buffer that inactivates RNAses and ethanol was subsequently added to provide suitable binding conditions. The samples were then added to RNeasy spin columns, allowing total RNA to bind to the membrane. Afterwards, contaminants were washed away and RNA was eluted in 50µl RNAse-free water. Lastly, RNA concentration and quality was assessed for all samples with a Denovix DS-11 FX+ spectrophotometer (Denovix, UK).
Label
biotin
Label protocol
Sense strand cDNA was generated, fragmented and labelled using the GeneChip WT Plus Reagent kit (Thermofisher UK) as per kit manufacturer directions.
Hybridization protocol
Samples were hybridised to the Clariom D GeneChip microarray (Thermofisher UK) as per directions for the GeneChip WT Plus Reagent kit (Thermofisher UK). Overnight hybridisation to the Clariom D GeneChip took place at 45oC for 16 hours.
Scan protocol
After washing and staining as per directions for the GeneChip WT Plus Reagent kit (Thermofisher UK) images were captured and cel files generated using the GeneChip Scanner 3000 and raw data converted to .CEL files using the Affymetrix GCOS Software (Thermofisher UK).
Description
mRNA expression data from Clariom D GeneChip
Data processing
The .CEL data were analysed using R/Bioconductor using an updated chip definition file (CDF) from the Brainarray resource based on Ensembl gene definitions (ENSG version 25.0.0). Raw microarray data were normalised using the SCAN method (Piccolo et al, 2012 DOI:10.1016/j.ygeno.2012.08.003). Normalised data were filtered using the UPC method (see Piccolo et al) with expression intensity and UPC filters were set at 0 and 0.5 respectively. Changes in differential expression were examined using empirical Bayes and a moderated t statistic implemented in the limma package. The SCAN algorithm generates a log2 signal for expression. The CDF used was the updated chip definition file (CDF) from the Brainarray resource based on Ensembl gene definitions (CDF = ENSG version 25.0.0)
