title: GSM7868517: rRNA3; Homo sapiens; OTHER
library_construction_protocol: The COMRADES assay was performed as described previously (Ziv et al., 2018). In brief, RNA was fragmented using RNase III (Ambion) and biotin was attached to crosslinked RNA duplexes using Click-IT Biotin sDIBO Alkyne (Life technologies). Biotinylated RNA duplexes were enriched using Dynabeads MyOne Streptavidin C1(Invitrogen) at the following reaction conditions: 100 mM Tris-Cl pH 7.5, 10 mM EDTA, 1 M NaCl, 0.1% Tween-20, 0.5 unit/µl Superase-In. Beads were washed 5 times on a magnet with 100 mM Tris-HCl pH 7.5, 10 mM EDTA, 3.5M NaCl, 0.1% Tween-20. RNA was eluted by adding 95% Formamide, 10 mM EDTA solution preheated to 65oC and purified using RNA Clean & Concentrator (Zymo Research). Each RNA sample was divided in two: one half was used for proximity ligation and then crosslink reversal (i.e. COMRADES sample), while in the other half, crosslink reversal was done before proximity ligation (i.e. control sample). Prior to proximity ligation, RNA was denatured by briefly heating to 95 °C. Proximity ligation was done under the following conditions: 1 unit/µl RNA ligase 1 (New England Biolabs), 1x RNA ligase buffer, 50 mM ATP, 1 unit/µl Superase-in (Invitrogen), final volume: 200 µl. Reaction was incubated for 16 hours at 16 °C and was terminated by cleaning with RNA Clean & Concentrator (Zymo Research). Crosslink reversal was done by irradiating the RNA on ice with 2.5KJ/m2 UVC. Library preparation was done as described in(Ziv et al., 2018), using 6N unique molecular identifiers to eliminate PCR biases. Pre-adenylated adapters were used and all ligation reactions were carried without ATP to reduce ligation artefacts. All libraries and controls went through 13 PCR cycles using KAPA HiFi HotStart Ready Mix (KAPA Biosystems). PCR products were size-selected on a 1.8% agarose gel before loading on a Novaseq (Illumina) for paired-end 150 bp runs