Gene Expression
Nebulas
Gene Expression NebulasSummary: Diminishing potential to replace damaged tissues is a hallmark for ageing of somatic stem cells, but the mechanisms leading to ageing remain elusive. We present a proteome-wide atlas of age-associated alterations in human haematopoietic stem and progenitor cells (HPCs) along with five other cell types that constitute the bone marrow niche. For each, the abundance of a large fraction of the ~12,000 proteins identified was assessed in a cohort of healthy human subjects from different age. As the HPCs became older, pathways in central carbon metabolism exhibited features reminiscent of the Warburg effect where glycolytic intermediates are rerouted towards anabolism. Simultaneously, altered abundance of early regulators of HPC differentiation revealed a reduced functionality and a bias towards myeloid differentiation at the expense of lymphoid development. Ageing caused significant alterations in the bone marrow niche too, such as functionality of the pathways involved in HPC homing and lineage differentiation. The data represents a valuable resource for further in-depth mechanistic analyses, and for validation of knowledge gained from animal models.
Overall Design: RNA-seq samples extracted from human bone marrow, from 6 cell populations (HPC, LYM, MON, ERP, GRA, MSC). Technical replicates are included for each donor and cell type. Technical replicates were produced by making independent libraries from the same RNA.
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| Growth Protocol: | Bone marrow (BM) samples were harvested through puncture at the posterior iliac crest using a Yamshidi needle |
| Treatment Protocol: | BM aspirates were processed by FICOLL density fractionation for isolation of MNCs; stained with CD34-APC, CD45-FITC and CD14-PE; FACS-sorted and stored at -80℃ |
| Extract Protocol: | RNA was extracted with trizol (Invitrogen) using a linear acrylamide carrier. RNA was then treated with DNase I (Life technologies) and purified using Agencourt RNAClean XP beads (Beckman Coulter). RNA quality and concentration were assessed by using an RNA 6000 bioanalyzer pico kit (Agilent). Samples with concentrations less than 30 pg/μl and/or an RNA integrity number (RIN) less than 6 were excluded from further analysis. |
| Library Construction Protocol: | A cDNA library was produced using the Smart-Seq 2 protocol. Sequencing was performed on an Illumina HiSeq4000 with 75 bp paired-end reads with the aim to achieve coverage of 25 million reads per sample. |
| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | PAIRED |
| Library Strand: | - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 4000 |
| Strand-Specific: | Unspecific |
| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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