biostudies-arrayexpressPeter SchjerlingHomo sapiensBBSplit, SubReadDESeq2bcl2fastqhttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-14395The study was designed to compare whether and how muscle fibroblasts (FIB) and muscle stem cells (MYO) interact with motor neurons. Moreover, we aimed to investigate whether muscle cells, isolated from lifelong exercisers, exerted a protective and supportive effect on motor neurons. These objectives were investigated in vitro using primary muscle cells from human donors and primary motor neurons from rat embryos and were analyzed by immunocytochemistry and species-specific RNA sequencing. A short period (24 hours) of co-culture was chosen for the RNA seq experiment, as differences on the gene expression level were expected to be greater initially. This accession contains the counts from the human RNA sequences.biostudies-arrayexpressLibrary Construction - RNA samples were quantified using Qubit 4.0 Fluorometer and RNA integrity was checked with RNA Kit on a Agilent 5300 Fragment Analyzer. RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina following manufacturer’s instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were first enriched with Oligo(dT) beads. Enriched mRNAs were fragmented according to manufacturer’s instruction. First strand and second strand cDNAs were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3’ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by limited-cycle PCR. Sequencing libraries were validated using NGS Kit on the Agilent 5300 Fragment Analyzer (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 4.0 Fluorometer (Invitrogen, Carlsbad, CA).Sequencing - The sequencing libraries were multiplexed and loaded on the flow cell on the Illumina NovaSeq 6000 instrument according to manufacturer’s instructions. The samples were sequenced using a 2x150 Pair-End (PE) configuration v1.5. Image analysis and base calling were conducted by the NovaSeq Control Software v1.7 on the NovaSeq instrument. Raw sequence data (.bcl files) generated from Illumina NovaSeq was converted into fastq files and de-multiplexed using Illumina bcl2fastq program version 2.20. One mismatch was allowed for index sequence identification. 17-77 million paired reads were obtained per sample.Sample Treatment - The human cells were acquired from sedentary young subjects or elderly that had performed life-long exercise or not.Sample Collection - Human fibroblasts and stem cells were released from Vastus lateralis muscle biopsies by enzymatic digestion and subjected to magnetic-activated cell sorting using a CD56 antibody to isolate CD56 positive cells (muscle stem cells) and CD56 negative cells (fibroblasts). Rat neuron cells were isoltaed from the spinal cord of E14 rat embryos. Dissociated cells were obtained by incubation of finely cut tissue with trypsin, followed by multiple trituration steps in DNase containing medium. Motor neuron enrichment at this stage was 3-5 %. The dissociated cells underwent density gradient centrifugation using Optiprep (D1556; Sigma-Aldrich), increasing the proportion of motor neurons to 30-40 %.Growth Protocol - Passage 0 human cells were thawed, cultured in growth medium for 5 days, and then plated at 10,000 cells/cm2, in 24-well plates containing coverslips (day -1). The next day rat neurons were added at 5,000 cells/cm2, and the medium was changed to a 1:1 mixture of differentiation medium (C-23260; PromoCell) with L-glutamine-penicillin-streptomycin solution (G6784; Sigma) and Neurobasal medium (A3582901; Gibco) with B27 supplement (A3582801; Gibco), L-Glutamine (25030032; Thermo Scientific) and three neurotrophic factors (GDNF (450-51; PeProTech), BDNF (450-02; PeProTech) and CNTF (450-50; PeProTech)). 24 hours later, experiments were stopped, and the cells were processed for RNA extraction.Nucleic Acid Extraction - Total RNA was isolated from the cell cultures using TriReagent.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Raw counts were normalized using the median ratio method (DESeq2)Sequence Alignment - The reads were split into Human and Rat reads using bbsplit (http://sourceforge.net/projects/bbmap/) by comparison to the Rat mRatBN7 and Human GRCh38 genomes. Ambiguous reads were excluded. Human and Rat reads were aligned to the respective genomes and transcripts (exons) counted using SubRead v2.0.3.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsQubit 4.0 Fluorometer/ Agilen 5300 Fragment AnalyzerIllumina NovaSeq 6000RNA-seq of coding RNAHomo sapiensMuscle fibroblasts and stem cells stimulate motor neurons in an age and exercise-dependent mannerCasper Soendenbroe, Peter Schjerling, Cecilie J. L. Bechshøft, Rene B. Svensson, Laurent Schaeffer, Michael Kjaer, Bénédicte Chazaud, Arnaud Jacquier, Abigail L. MackeyPeter SchjerlingfalseHuman skeletal muscle fibroblasts and stem cells cultured with rat motor neuron cellsThe study was designed to compare whether and how muscle fibroblasts (FIB) and muscle stem cells (MYO) interact with motor neurons. Moreover, we aimed to investigate whether muscle cells, isolated from lifelong exercisers, exerted a protective and supportive effect on motor neurons. These objectives were investigated in vitro using primary muscle cells from human donors and primary motor neurons from rat embryos and were analyzed by immunocytochemistry and species-specific RNA sequencing. A short period (24 hours) of co-culture was chosen for the RNA seq experiment, as differences on the gene expression level were expected to be greater initially. This accession contains the counts from the human RNA sequences.2026-02-21T00:00:00Z2026-02-21T02:02:32.535Z2024-08-30T16:02:58.879ZE-MTAB-14395E-MTAB-14397E-MTAB-14399EFO_0002944EFO_0004170EFO_0003789EFO_0004917EFO_0005518EFO_0003816EFO_0003738EFO_0004184EFO_0003969https://onlinelibrary.wiley.com/doi/10.1111/acel.14413