title: GSM3753373: 293T_ILF2OE_1; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was extracted from cells using Qiagen Micro or Mini RNeasy kit (Qiagen cat:74004 or 74104) using RNAdvanced Tissue Kit (Beckman Coulter) followed by TURBO DNase treatment (Thermo Fisher). rRNA was depleted from total RNA following RNase H-based protocols adopted from (Adiconis et al., 2013; Morlan et al., 2012). 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 5ul reaction with 1 ul of 5X RNase H(-)Mg buffer (500 mM Tris-HCl pH 7.4, 1 M NaCl) and 0.25 ul 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 ul of RNase H mix (1.7 ul 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 ul of Hybridase Thermostable RNase H (Epicenter, Madison, WI: Lucigen H39500) to make 10 ul 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).