GSM8522014: RMS_ESC_A; Mus musculus; OTHER
Total RNA was extracted using Trizol. 150 to 250 ng of DNase-treated RNA was hydrolyzed in 50 mM sodium bicarbonate buffer (pH 9.2) for 1 to 12 minutes. The hydrolysis reaction was quenched in 1 mL EtOH containing 10 uL 3 M NaOAc, pH 5.2 (Thermo) and 15 ug Glycoblue coprecipitant (Thermo) and submerged in liquid nitrogen. Hydrolyzed RNA was then precipitated and washed in 80% EtOH. The intended fragment size of around 50-100 bp was verified on the TapeStation 4200 system (Agilent). RNA was dephosphorylated by Antarctic Phosphatase (New England Biolabs) for 30 minutes at 37°C, and end-repaired by T4 PNK (New England Biolabs) in the presence of 1 mM ATP (New England Biolabs) for 1 hour at 37°C. End-repaired RNA was purified using RNA Clean & Concentrator-5 columns (Zymo). Illumina libraries were then prepared using the NEBNext® Multiplex Small RNA Library Prep Set for Illumina® kit (New England Biolabs) and purified on NucleoSpin® Gel and PCR Clean-up columns (Takara Bio).