Zymo rna clean and concentrator

In vitro transcription reactions were performed using PCR products generated with primers encoding a flanking T7 RNA polymerase promoter and a poly-A tail. Reactions were set up, as previously described (66 (link)), with 20 mM Tris-HCl pH 7.5, 35 mM MgCl₂, 2 mM spermidine, 10 mM DTT, 1 u/ml pyrophosphatase (Sigma), 7.5 mM of each NTP, 0.2 u/ml RiboLock RNase Inhibitor (ThermoFisher), 0.1 mg/ml T7 RNA polymerase and 40 ng/μl PCR-generated DNA. After 3 h incubation at 37 °C, 0.1 u/μl DNase I (Promega) was added to the reactions, which were incubated at 37 °C for 30 min to remove the template DNA. RNA was precipitated for 2–3 h at −20 °C after adding 0.5x volume of 7.5 M LiCl/50 mM EDTA, and the resulting pellet was washed with cold 70% ethanol and dissolved with RNase-free water. The mRNA was further purified by using a Zymo RNA Clean and Concentrator (Zymo Research) before use in in vitro translation reactions.
DNA templates were amplified from a plasmid containing the corresponding 5’ UTR and the NanoLuc Luciferase coding sequence. Primers used for this amplification added a 30T sequence at the 3′ end to form a poly(A) tail after transcription. The HBB 5’ UTR containing mRNA was then capped using Vaccinia Capping enzyme (New England Biolabs) and 2′O-methylated using Vaccinia 2′O Methyltransferase (New England Biolabs). The IRES-containing mRNAs were uncapped and polyadenylated.

The samples used were from a previously published study (Yeri et al., 2017 (link)). Plasma and saliva samples were isolated (1 ml) using the mirVana miRNA Isolation Kit (ThermoFisher Scientific, Waltham, MA, AM1560) according to (Burgos et al., 2013 (link)). Samples were DNase treated using TURBO DNA-free Kit (ThermoFisher Scientific, Waltham, MA, AM 1907). Samples were then cleaned and concentrated using Zymo RNA Clean and Concentrator (Zymo Research, Irvine, CA, R1016) using Protocol: Purification of small and large RNAs into separate fractions and combining the fractions at the end. Urine samples (15 ml) were isolated with Urine Total RNA Purification Maxi Kit, Slurry Format (Norgen Biotek Corp., Thorold, ON, Canada, Cat#29600). Samples were DNase treated on column using RNase-Free DNase Set (Qiagen, Germantown, MD, cat# 79254). Samples were concentrated by Speed Vacuum.

The 5′UTRs of reporter RNA were synthesized by GeneArt (Thermo Fisher Scientific). Templates were prepared by Fusion PCR using the 5′UTR variants, ORFs and common 3′UTR with a poly-A tail. Capped mRNAs were prepared by using the MegaScript Kit (Thermo Fisher Scientific) in the presence of an anti-reverse cap analog (TriLink BioTechnologies). Ten microliters of the reaction solution was prepared and incubated at 37 °C. After 6 h of incubation, 1 μL of TURBO DNase (Thermo Fisher Scientific) was added to the reaction solution, mixed by pipetting, and incubated at 37 °C for 30 min. RNA products were purified with Zymo RNA Clean and Concentrator (Zymo Research). Then, to remove the phosphate at the 5′ terminus, the RNA products were treated with Antarctic Phosphatase (New England Biolabs). The RNA products were purified again, and the concentrations were measured with a NanoDrop 2000 (Thermo Fisher Scientific). Sequence information regarding all DNA materials for constructing the templates of reporter mRNA is available in Supplementary Data 1.

RNA was DNase I-treated, cleaned and concentrated (Zymo RNA Clean and Concentrator, Zymo Research) then enriched for poly(A) mRNA (NEBNext poly(A) mRNA Magnetic Isolation Module, NEB Biosystems, Ipswich, UK). Sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep kit (New England Biolabs). RNA and DNA quality were assessed using High Sensitivity RNA or DNA Screentape and an Agilent 4200 tapestation. Single-indexed and multiplexed samples were run on an Illumina Next Seq 500 sequencer using a NextSeq 500 v2 kit (FC-404–2005; Illumina, Can Diago, CA) for paired-end sequencing.
A computational pipeline was written calling scripts from the CGAT toolkit (https://github.com/cgat-developers/cgat-flow)^{39 (link),40 (link)}. Sequencing reads were de-multiplexed based on the sample index and aligned to the human genome assembly version 38 (GRCh38) using the STAR (Spliced Transcripts Alignment to a Reference) aligner^{41 (link)}. At least 14 million aligned reads were obtained per sample. Reads were mapped to genes using featureCounts v1.4.6 (part of the subreads package), in which only uniquely mapped reads were counted to genes. Differential expression analysis was performed using DESeq2^{42 (link)} between three groups: osteoclasts cultured on cell culture plastic, dentine or cellular cartilage.