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| Status |
Public on Jun 14, 2023 |
| Title |
250718B_IL1B |
| Sample type |
SRA |
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| Source name |
Superficial digital flexor tendon tenocytes
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| Organism |
Equus caballus |
| Characteristics |
tissue: Superficial digital flexor tendon tenocytes
genotype: WT
treatment: Interleukin 1 beta
batch: First
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| Treatment protocol |
Tenocytes were seeded in type 1 collagen gels (bovine; Advanced Biomatrix, California, USA) to generate a three-dimensional matrix and stimulated with IL-1ß (1 nM), IL-1ß (1 nM) + IL1Ra (100 ng/mL) or control for 14 days, with fresh media changes every 3-4 days before harvesting at day 14.
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| Growth protocol |
Adult tenocytes were cultured in Dulbecco’s modified eagle medium (DMEM) high glucose (4.5 g/L), 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin (all from Invitrogen, Renfrewshire, UK) and passaged using 0.25% trypsin-EDTA (Sigma-Aldrich) every 3-4 days.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using Tri-reagent (Sigma), purified using an RNeasy mini Kit (Qiagen, Manchester, UK) and contaminating genomic DNA removed using Ambion DNA-free (Life Technologies, Paisley, UK). RNA concentration was measured using a Nanodrop (ThermoFisher, Loughborough, UK) and a Qubit (ThermoFisher). RNA integrity was confirmed on a Tapestation (Agilent, Milton Keynes, UK).
mRNA libraries were prepared using a TruSeq stranded mRNA kit (Illumina, Cambridge, UK).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
250718BIL1B
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| Data processing |
RNA sequencing was performed on a NovaSeq6000 (Illumina) by external providers (Oxford Genomics, Oxford, UK & Novagene, Cambridge, UK).
30.7 – 46.2 million reads of 150 base pair paired-end data were generated per sample.
Resulting FASTQ files generated were quality control (QC) checked using FASTQC and MultiQC (Babraham Bioinformatics, Cambridge, UK).
QC checked reads were then aligned to the Ensembl version v95 EquCab 3.0 transcriptome using the pseudoaligner Salmon in Quasi-mapping based mode with GC-bias correction (-gcBias).
The tximport package (v.1.24; Soneson et al. 2015) was used to import the quantified gene-level abundance data into R (v.4.2.1) and differential expression analysis conducted using R/Bioconductor DESeq2 (v.1.36) as described in Love et al., 2014.
Assembly: Genes with adjusted pvalue of less than 0.05 and log2 fold change of ± 2 were considered as differentially expressed.
Supplementary files format and content: Ensembl version v95 EquCab 3.0.
Supplementary files format and content: Control_vs_IL1B_First_Batch_Count_Data.csv: Comma-Separated Value (CSV) files include raw counts of sequencing reads for each sample from the first batch (n=5 biological replicates, two conditions).
Supplementary files format and content: Control_vs_IL1B_vs_IL1Ra_Second_Batch_Count_Data.csv: Comma-Separated Value (CSV) files include raw counts of sequencing reads for each sample from the second batch (n= 3 biological replicates, three conditions).
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| Submission date |
Dec 19, 2022 |
| Last update date |
Jun 14, 2023 |
| Contact name |
Ross Eric Beaumont |
| E-mail(s) |
robeaumont@rvc.ac.uk
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| Phone |
07312130009
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| Organization name |
Royal Veterinary College
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| Department |
Clinical science and services
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| Lab |
Skeletal biology group
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| Street address |
Hawkshead Ln
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| City |
Hatfield |
| State/province |
Hertfordshire |
| ZIP/Postal code |
AL97TA |
| Country |
United Kingdom |
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| Platform ID |
GPL26749 |
| Series (1) |
| GSE221370 |
Exogenous interleukin-1 beta stimulation regulates equine tenocyte function and gene expression which can only be rescued by pharmacological inhibition of interleukin 1 receptor, but not nuclear factor kappa B, signalling |
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| Relations |
| BioSample |
SAMN32316917 |
| SRA |
SRX18782074 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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