biostudies-arrayexpressPeter SchjerlingRattus norvegicusBBSplit, SubReadDESeq2bcl2fastqhttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-14399The 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. Rat neuron cells were treated for 24 hours with conditioned media from human skeletal muscle fibroblasts and myoblasts.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).Nucleic Acid Extraction - Total RNA was isolated from the cell cultures using TriReagent. 400 ng total RNA purified using TriReagent from bakers yeast (Frisk Bagegær, De Danske Gærfabrikker, Grenaa, Denmark) was added to the TriReagent to minimize loss of rat neuron RNA due to the very low amount.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 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 isolated 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 (fibroblasts or myoblasts) were cultured for 7 days in growth medium (cat. no. C-23060; PromoCell and supplement mix cat. no. C-39365) containing 15% fetal bovine serum (FBS; cat. no. ALB-S1810; Biowest) and 5 μl of l-glutamine-penicillin-streptomycin solution (200 mM l-glutamine, 10 mg/ml streptomycin, 10,000 units penicillin, cat. no. G6784; Sigma). During the incubation the medium was replaced twice at day 2 and day 5. The day 5 and 7 media frozen down as \\"conditioned\\" medium. Motor neurons were plated at 5,000 cells/cm2 in 24-well plates on coverslips coated with Poly-L-Ornithine (P8638; Sigma-Aldrich) and Laminin (L2020; Sigma-Aldrich). The conditioned medium was centrifuged to remove cell debris. The neurons were plated in 60 % Neurobasal medium (A3582901; Gibco) and 40 % conditioned medium from the human cells. 24 hours later, experiments were stopped and processed for RNA extraction.Sample Treatment - The rat neuron cells were cultured with conditioned meium from either human firbroblasts or myoblasts.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Rat raw counts were normalized using the median ratio method (DESeq2)Sequence Alignment - The reads were split into Rat and Yeast reads using bbsplit (http://sourceforge.net/projects/bbmap/) by comparison to the Rat mRatBN7 and Saccharomyces cerevisiae R64 genomes. Ambiguous reads were excluded. 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 RNARattus norvegicusMuscle 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 SchjerlingfalseEffects of human skeletal muscle fibroblast and stem cell media on cultured 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. Rat neuron cells were treated for 24 hours with conditioned media from human skeletal muscle fibroblasts and myoblasts.2026-02-21T00:00:00Z2026-02-21T02:02:32.522Z2024-08-30T16:03:46.922ZE-MTAB-14399E-MTAB-14395E-MTAB-14397EFO_0002944EFO_0004170EFO_0003789EFO_0004917EFO_0005518EFO_0003816EFO_0003738EFO_0004184EFO_0003969https://onlinelibrary.wiley.com/doi/10.1111/acel.14413