Rnaclean xp beads

rRNA was depleted from total RNA following RNase H-based protocols adopted from Adiconis et al. (2013) (link) and Morlan et al. (2012) (link). We mixed approximately 150 ng of RNA with 150 ng of pooled DNA oligos designed antisense to Drosophila rRNA in 50 base pair sections (Table S5) in an 8 μL reaction with μL of 5X Hybridization buffer (500 mM Tris-HCl pH 7.4, 1 M NaCl). We annealed rRNA antisense oligos to total RNA samples for 2 minutes at 95°C, slowly reduced the temperature to 45°C and then added 2U of Hybridase Thermostable RNase H (Epicenter, Lucigen: H39500) and 1 μL of 10X Digestion buffer (500 mM Tris-HCl, 1 M NaCl, 200mM MgCl₂) and incubated for 30 minutes at 45°C. rRNA-depleted RNA was then purified using 2.2X reaction volume of Agencourt RNAClean XP beads (Beckman Coulter: A63987), treated with TURBO DNase (Invitrogen: AM1907), and then purified with RNAClean XP beads again. rRNA-depleted RNA was used as input to the KAPA Stranded RNA-seq Kit (Kapa Biosystems: KK8400) to make RNA-sequencing libraries for fly knockdowns. For mouse primary neuron RNA-seq libraries, the KAPA HyperPrep RNA-seq Kit (Kapa Biosystems: KK8540) was used to create libraries after rRNA depletion using oligos antisense to human rRNA sequences (Adiconis et al., 2013 (link)). All libraries were sequenced with 76 base pair paired-end reads using an Illumina NextSeq.

Total RNA was extracted from cells using QIAGEN Micro or Mini RNeasy kit (QIAGEN cat:74004 or 74104). rRNA was depleted from total RNA following RNase H-based protocols adopted from Adiconis et al. (2013) (link) and Morlan et al. (2012) (link). We mixed approximately 250 ng of RNA with 61.54 pmoles of pooled DNA oligos designed antisense to rRNA (gift from J. Salzman lab at Stanford) in a 5 μL reaction with 1 μL of 5X RNase H(−)Mg buffer (500 mM Tris-HCl pH 7.4, 1 M NaCl) and 0.25 mL 1mM EDTA. We annealed rRNA antisense oligos to total RNA samples for 2 minutes at 95°C, slowly reduced the temperature to 45°C and then added 5 μL of RNase H mix (1.7 μL water, 1 ul 5X RNase H(−)Mg buffer, 0.2 ul 1 M MgCl₂, 0.1ul RiboLock RNase Inhibitor (40 U/μL) (ThermoFisher EO038), 2 μL of Hybridase Thermostable RNase H (Epicenter, Madison, WI: Lucigen H39500) to make 10 μL total and incubated for 30 minutes at 45°C. rRNA-depleted RNA was then purified using 2.2X reaction volume of Agencourt RNAClean XP beads (Beckman Coulter: A63987), treated with TURBO DNase (Invitrogen: AM1907), and then purified with RNAClean XP beads again. rRNA-depleted RNA was used as input to the KAPA HyperPrep RNA-seq Kit (Kapa Biosystems: KK8540). All libraries were sequenced with 76 base-pair paired-end reads using an Illumina NextSeq (Illumina, San Diego, CA).