title: GSM2428798: 4SU-MagoKD_t0_Rep2; Drosophila melanogaster; RNA-Seq
library_construction_protocol: 5,6-dichlorobenzimidazole 1-_-d-ribofuranoside (DRB) from Sigma (D1916) was used at a final concentration of 300_M, dissolved in water, for 5 hours. 4-thiouridine (4sU) was purchased from Sigma (T4509) and used at a final concentration of 100 _M. Control and Mago KD was performed as described before. All the samples were labeled for 8 minutes with 4-thiouridine, and transcription was allowed to proceed after DRB removal for 0, 2, 8 and 16 minutes along with one non-DRB treated control.A total of 100 to 130 ug RNA was used for the biotinylation reaction. 4sU-labeled RNA was biotinylated with EZ-Link Biotin-HPDP (Pierce), dissolved in dimethylformamide (DMF) at a concentration of 1 mg/mL. Biotinylation was done in labeling buffer (10 mM Tris pH 7.4, 1 mM EDTA) and 0.2 mg/mL Biotin-HPDP for 2 h with rotation at room temperature. Two rounds of chloroform extractions removed unbound Biotin-HPDP. RNA was precipitated at 20,000 g for 20 min at 4¡C with a 1:10 volume of 5M NaCl and an equal volume of isopropanol. The pellet was washed with 75% ethanol and precipitated again at 20,000 g for 10 min at 4¡C. The pellet was left to dry, followed by resuspension in 100 _L RNase-free water. Biotinylated RNA was captured using Dynabeads MyOne Streptavidin T1 beads (Invitrogen). Biotinylated RNA was incubated with 50 _L Dynabeads with rotation for 15 min at 25¡C. Beads were magnetically fixed and washed with 1_ Dynabeads washing buffer. RNA-4sU was eluted with 100 _L of freshly prepared 100 mM dithiothreitol (DTT), and cleaned on RNeasy MinElute Spin columns (Qiagen). Enriched nascent RNAs were converted to cDNA libraries with Drosophila Ovation Kit (Nugen) with integrated ribosomal depletion workflow
attributes.tag: GEO Accession
attributes.value: GSM2428798