GSM5861362: TTseq_GM10253A_ExoKD_1; Homo sapiens; Cricetulus griseus; RNA-Seq
RNA extraction was performed with TRIzol (Invitrogen) following the manufacturers' instructions. After DNase treatment (Turbo DNase, Thermo Fisher Scientific) RNA concentration and purity were determined using a NanoDrop. RNA (70 μg) was fragmented in 100 μl H20 to <1.5kb by 20 cycles of 30 seconds on/30 seconds off at high power in a Biorupter plus and RNA size assessed by agarose gel electrophoresis. Fragmented 4sU labelled RNA was biotinylated by adding 140 μl of EZ-Link Biotin-HPDP (1mg.ml-1 in dimethylformamide; Pierce, 21341), 70 μl of 10 x biotinylation buffer (100 mM Tris-HCl pH 7.5, 10 mM EDTA) and H20 to a final volume of 700 μl. This was incubated at room temperature for 1.5 h with rotation. Unincorporated biotin-HPDP was removed by two rounds of chloroform extraction with 2 ml Phase lock gel heavy tubes (Eppendorf). RNA was precipitated with 1/10 volume of 5M NaCl and an equal volume of Isopropanol. This was inverted to mix and incubated at room temperature for 10 min followed by centrifugation at 10,000g for 20 min at room temperature. RNA pellet was washed with 80% EtOH and centrifuged at 13,000 rpm for 10 min at 4°C. RNA was resuspended in 100 μl H20 and dissolved by heating to 40°C for 10 min with agitation. RNA was then immediately placed on ice and RNA concentration determined by Nanodrop spectrophotometer. Biotinylated 4sU labelled RNAs were then recovered using μMACS Streptavidin MicroBeads (Miltenyi, 130-074-101) and separation on a μMACS Separator. For the concentration of total RNA in μg per sample an equal amount in μl of Streptavidin microbeads was added. This was incubated at room temperature for 15 min with rotation. μMacs columns were equilibrated with 900 μl room temperature washing buffer (100 mM Tris-HCl pH 7.5, 10 mM EDTA, 1 M NaCl, 0.1% Tween20). The RNA/streptavidin bead solution was then applied to the column followed by three washes with 900 μl of washing buffer at 65°C and three washes with 900 μl of washing buffer at room temperature. RNA was eluted with 2 x 100 μl of fresh elution buffer (100 mM dithiothreitol in RNase-free H20 ) directly into 2 ml lobind tubes (Eppendorf) containing 700μl Buffer RLT (RNeasy MinElute Cleanup Kit, Qiagen). 500 μl of 100% ethanol was added to the RNA solution, and mixed thoroughly by pipetting before RNA was purified through RNAeasy MinElute Spin Columns. RNA concentration was determined using a Nanodrop. Libraries for RNA-seq were prepared and indexed using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (NEB #E7645L) and NEBNext Singleplex Oligos for Illumina (NEB #E7335, E7500) following the manufacturer's instructions. 