title: GSM3610101: Zn72D-GFP_input_1; Drosophila melanogaster; RIP-Seq
library_construction_protocol: RNA immunoprecipitation was performed after homogenizing ~500 ul of fly heads in IP Buffer (150 mM NaCl, 20 mM HEPES pH 7.5, 2 mM MgCl2, 0.1% NP40, cOmplete protease inhibitor, RNaseOUT RNase inhibitor 1U/ul (Invitrogen)). Lysates were split in half, and 4% of input was removed for input control libraries. IP lysates were incubated overnight at 4C with Dynabeads Protein G (Invitrogen: 10003D), plus 5 ug of anti-GFP antibody (Abcam: ab290). IPs were washed 8 times in IP buffer. Beads with protein bound and saved inputs were added to 1 mL of TRIzol (Thermo Fisher: 15596026). 200 ul of chloroform was added, and samples were centrifuged at 14000 xg at 4C for 15 minutes. Aqueous phases were collected, mixed with 1 volume of 70% ethanol and then transferred to a RNeasy MinElute column (Qiagen, Hilden, Germany: 74204) for purification following the standard protocol. RNA was mixed with an equal amount of pooled DNA oligos designed antisense to Drosophila rRNAs in 50 bp sections in an 8 ul reaction with 2ul of 5X Hybridization buffer (500 mM Tris-HCl pH 7.4, 1  M NaCl). rRNA antisense oligos were annealed to total RNA samples for 2 minutes at 95°C, the temperature was slowly reduced to 65°C and 2U of Thermostable Hybridase H (Epicentre) and 1 ul of 10X Digestion buffer (500 mM Tris-HCl, 1 M NaCl, 200mM MgCl2) were added to make 10 ul total and reactions were incubated for 30 minutes at 65°C. rRNA-depleted RNA was then purified using Agencourt RNAClean XP beads, treated with TURBO DNase (Ambion) and then purified with RNAClean XP beads again. rRNA-depleted RNA was used as input to the KAPA HyperPrep RNA-seq Kits (Kapa Biosystems: KK8540) to make RIP-seq libraries for fly samples.
identifiers.namespace: GEO
identifiers.id: GSM3610101