GSM5703117: BC_8; Mus musculus; RNA-Seq
After euthanasia, tibiae from both hindlimbs of the mice were dissected, measured and weighed. Then, we collected the proximal epiphysis of each tibia (consisting of the growth plate, metaphysis, articular cartilage and secondary ossification center), which were snap-frozen in liquid nitrogen. Before total RNA extraction, the frozen epiphyses were homogenized using a ball mill (Braun Mikro-Dismembrator S) at 2600rpm for 60 seconds. Total RNA-Seq Kit (Life Technologies) were used to obtain Poly-adenylated mRNAs only from the total RNA. PolyA RNA was converted into bar-coded cDNA libraries and size selected using Agencourt AMPure XP beads and then amplified using PCR to obtain around 50ng of 240-270 base pairs length cDNAs Equal amounts of each library were pooled together. Using the EZ-Bead system (Life Technologies) magnetic sequencing beads were added to the libraries. A yield of 2.2 billion beads was obtained. The samples were then sequenced on a 5500xl Genetic Analyzer (Life Technologies) to generate 35-50 base paired-end reads