Illumina Genome Analyzer sequencing; Differential expression of snoRNAs during ATDC5 chondrogenic differentiation
Differential expression of snoRNAs during ATDC5 chondrogenic differentiation
Cells were harvested for RNA isolation with TRIzol reagent (Invitrogen).  ATDC5 cells (RIKEN BRC, Japan) were cultured in a humidified atmosphere at 37oC, 5% CO2 in proliferation medium (DMEM/F12 (Invitrogen, Carlsbad, CA, USA), 5% FCS (Sigma-Aldrich, St. Louis, MO, USA) and 1% antibiotic/antimycotic (Invitrogen). Cells were plated at 6,400 cells/cm2. After 24 hours chondrogenic differentiation was initiated by changing the medium to differentiation medium (proliferation medium supplemented with 10 μg/ml insulin (Sigma-Aldrich), 10 μg/ml transferrin (Roche, Basel, Switzerland) and 30 nM sodium selenite (Sigma-Aldrich)). Differentiation medium was refreshed every two days for the first 10 days, and each day after day 10, until cells were harvested at indicated time points. RNA was isolated from the TRIzol samples by collecting the aqueous phase after phase separation. RNA was precipitated with isopropanol (30 minutes, -80oC) and centrifuged for 30 minutes at 20,000 x g, 4oC. RNA pellets were washed with 80% ethanol and potential DNA contamination was removed by DNase I (Roche) treatment (1 hour, 37oC). After subsequent ethanol precipitation, RNA was dissolved in 15 μL DNase/RNase free water (Eurogentec, Seraing, Belgium). RNA quantity and purity were determined spectrophotometrically (Biodrop, Isogen Life Sciences, Utrecht, the Netherlands).  SnoRNA expression during ATDC5 differentiation was analyzed by small RNA Next Generation Sequencing (NGS) of samples acquired from day 0 (progenitor stage), day 7 (chondrogenic stage) and day 14 (hypertrophic stage). Total RNA was isolated and after library preparation (NEBNext library generation kit, New England Biolabs (NEB), Ipswich, MA, USA) inserts <200 nt were selected using an automated gel cutter. One µg of total RNA was converted into NGS libraries using the NEBNext library generation kit (NEB), using a protocol (Exiqon, Vedbæk, Denmark) to increase the 5' and 3' ligation of modified small RNA ends. Each individual RNA sample had adapters ligated to its 3' and 5' ends and was converted to cDNA. Then, the cDNA was pre-amplified with specific primers containing sample specific indexes (Exiqon). After a 15 cycle pre-PCR the libraries were purified on QiaQuick column (Qiagen, Hilden, Germany) and the insert efficiency was evaluated on a Bioanalyzer 2100 instrument (Agilent, Santa Clara, CA, USA) on a high sensitivity DNA chip (Agilent). The cDNA libraries were size fractioned on a LabChip XT (Caliper Life Sciences, Waltham, MA, USA) and 15-200 bp inserts were excised according to the manufacturer's protocol (Caliper). Samples were then quantified using qPCR and a concentration standard. Based on the quality of the inserts and the concentration measurements the libraries were pooled in equimolar concentrations. The library pool was quantified with qPCR and an optimal concentration of the library pool (Illumina) was used to generate the clusters on the surface of a flowcell before sequencing (using v3 sequencing methodology according to the manufacturer's instructions).