GSM2936686
GSM2936686: 0.01d_37T_biotinylated; synthetic construct; OTHER
The cells were washed with PBS, trypsinized and suspended in 20 mL of supplemented MEM Earl´s. The suspension was centrifuged (800 rpm, 10 min), the medium was removed and the pellet was stored at -80 °C. Cell pellets from HeLa cells (~2.5 – 3.0•105 cells) were lysed mechanically by pipetting up and down in 5 mL lysis buffer (10 mM TrisHCl (pH = 8.0), 150 mM NaCl, 0.5 mM NP40, adjusted to pH 7.5 with HCl). Total RNA was purified by two consecutive phenol-chloroform extractions (4:1 and 2:1) followed by back-extraction with NaCl (0.9 %) and EtOH precipitation. The resulting total RNA was treated with 1 U DNase I (Thermo Fisher) in 1× DNase buffer (Thermo Fisher) for 30 min at 37°C, followed by phenol-chloroform (5:1) extraction and EtOH precipitation. For sequencing of total RNA/rRNA, total RNA was digested with 44 mM NaHCO3, 6 mM Na2HCO3 and 1 mM EDTA for 20 min at 95°C. The samples were precipitated with ethanol, followed by separation via denaturing PAGE and SYBRGold staining. The region from 50–80 nt was excised, RNA was eluted and precipitated. For total RNA and synthetic oligos RNA was dephosphorylated at the 5′-end using T4 PNK  (Thermo Scientific), followed by either magnetic separation and washing with ddH2O (biotinylated RNA) or phenol/chloroform extraction and precipitation (unmodified RNA). Ligation of a short, 5'-adenylated linker (40 ng/µL, NGS 4N linker) was performed with T4 RNA Ligase 2(truncated K227Q) (NEB) . Again, RNA was purified by magnetic separation and washing with ddH2O (biotinylated RNA) or phenol/chloroform extraction and precipitation (unmodified RNA). For the RT, biotinylated RNA remained on the magnetic streptavidin beads. To this end, 1 µM NGS RT-primer was hybridized and incubated with Maxima H Minus. After RT, template RNA was degraded by alkaline hydrolysis (125 mM NaOH at 80 °C, 10 min) before neutralizing with HCl. The obtained cDNA was separated via denaturing PAGE (10 % PAA gel, 8 M urea, 1× TBE) and visualized with SYBRGold staining. The region from 130–200 nt was excised, cDNA was eluted and precipitated. The purified cDNA was circularized using CircLigase II (Epicentre).  Circularized cDNA was then amplified with primers introducing an index to label samples for sequencing, and purified by native PAGE.
GEO
GSM2936686
GEO Accession
GSM2936686