title: Illumina HiSeq 2000 paired end sequencing; RNASeq of musculoskeletal ageing using human mesenchymal stem cells and their tissue constructs
design: RNASeq of musculoskeletal ageing using human mesenchymal stem cells and their tissue constructs
library_construction_protocol: MScs purchased from young and old human donors miRNAeasy kit for RNA extraction Total RNA samples were ribosomal RNA depleted using the Human, mouse, rat low inputRibozero kit from Epicentre. 1ug of RNA (or the max amount available) was mixed with the low input kit baits and allowed to anneal. This was then mixed with the magnetic microspheres which bind the ribosomal complexes and subsequently pulled out with a magnet. The remaining RNA was purified with RNA clean beads and analysed with the Agilent Bioanalyser with a RNA pico chip. Recovery was generally less than 1% of the original starting material. All of this material was used in the script seq reaction (Epicentre). Samples were denatured and annealed to cDNA primers and cDNA was generated. This was amplified with indexed primers for 15 cycles to make the library for sequencing. Libraries were checked on the Agilent Bioanalyser with a High sensitivity chip and quantities determined with Qubit DNA High sensitivity reagents.
attributes.tag: Experimental Factor: tenocyte
attributes.value: developmental stage
attributes.tag: Experimental Factor: cell
attributes.value: cell type