title: GSM3610066: Dmel_Irp-1A_RNAi_2; Drosophila melanogaster; RNA-Seq
library_construction_protocol: RNA was extracted from ~15 brains using RNAdvanced Tissue Kit (Beckman Coulter) followed by TURBO DNase treatment (Thermo Fisher). 150-250 ng of total 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 Stranded RNA-seq Kit (KK8400) to make RNA-sequencing libraries.
identifiers.namespace: GEO
identifiers.id: GSM3610066