Ribominus eukaryote kit

mRNA was isolated from total RNA by using a Dynabeads mRNA Purification Kit (Thermo Fisher Scientific), and rRNA contaminants were removed by using a RiboMinus Eukaryote Kit (Thermo Fisher Scientific). Subsequently, mRNA was digested into nucleosides by using nuclease P1 and alkaline phosphatase and was then filtered with a 0.22 mm filter. The amount of m⁶A was measured according to HPLC–tandem mass spectrometry, following the published procedure [26 (link)]. Quantification was performed by using the standard curve obtained from pure nucleoside standards that were run with the same batch of samples. The ratio of m⁶A to A was calculated based on the calibrated concentrations.

Ribosomal RNA was removed from samples using RiboMinus Eukaryote Kit (Thermo Fisher Scientific), and libraries for mRNA-seq were prepared using Ion Total RNA-seq Kit v2 (Thermo Fisher Scientific). The sizes of fragmented RNA and library cDNA were determined using the MultiNA microchip electrophoresis system (Shimadzu), and the quantity of library cDNA was determined by qPCR using KAPA Library Quantification Kit (KAPA Biosystems). mRNA-seq was performed using the Ion Proton sequencer and Ion PI Chip v3 (Thermo Fisher Scientific). Mapping of sequenced reads to the human genome reference hg19 and calculation of fragments per kilobase of transcript per million mapped reads (FPKM) were performed using the software CLC Genomic Workbench (Qiagen). All data were deposited in NCBI’s Gene Expression Omnibus and are publicly accessible through GEO accession number GSE120734. Fold change of gene expression was calculated using the following equation. DAVID 6.8 was used for gene ontology (GO) analysis. $$\mathrm{Fold}\mathrm{change}=({\mathrm{FPKM}}_{\mathrm{etoposide}}+1)/({\mathrm{FPKM}}_{\mathrm{control}}+1)$$

Ribosomal RNA depleted RNA libraries were generated and sequenced on a SOLiD5500 platform (Nord University, Bodø, Norway) as described in detail previously (24 (link)). For each cell line, a single biological replicate was used to generate one RNA-Seq library. The RiboMinus™ Eukaryote Kit (ThermoFisher Scientific) was used for rRNA depletion, and the libraries were prepared according to the SOLiD™ Total RNA-Seq Kit Protocol by Thermo Fisher Scientific. Adaptor-trimmed sequencing reads were mapped to the human genome (forward specific mapping, human reference sequence GRCh38.104) using the CLC Genomic Workbench 22. Normalized gene expression values (counts per million, CPM) were log2 transformed and calculation of significantly differentially expressed genes were done in CLC. RNA-Sequencing raw reads have been deposited to SRA (PRJNA976177). Analyzed data (full gene list) can be found in Supplementary Table 1. Publicly available datasets were downloaded from SRA, using the SRAtoolkit.

We used Ewing sarcoma cells (HTB166) as representative sarcoma samples and white blood cells from a healthy volunteer as control samples. Total RNA was extracted using an RNeasy^® Mini Kit (Qiagen, Hombrechtikon, Switzerland), and rRNA was deleted using the RiboMinus Eukaryote Kit (Thermo Fisher Scientific, Waltham, MA, USA). Whole-transcriptome sequencing was performed using the Ion Torrent Next-Generation Sequencing System (Thermo Fisher Scientific).

Total RNA was extracted from whole cells using TRIzol reagents (Thermo Fisher Scientific) following the manufacturer’s instructions.
To isolate the caRNA, the chromatin pellet was first obtained as described in the ‘Cell fractionation’ section. The pellet was then submerged in the TRIzol reagents and heated at 50 °C with shaking at 1,000 rpm for 1–2 h until the pellet completely dissolved. The caRNA was extracted according to the manufacturer’s instructions for TRIzol reagents.
To remove rRNA from whole cellular total RNA or caRNA, RiboMinus Eukaryote kit (Thermo Fisher Scientific) was used following the manufacturer’s protocol. For extraction of polyadenylated RNA from total RNA, the Dynabeads mRNA DIRECT kit (Thermo Fisher Scientific) was used according to the manufacturer’s instructions. The RNA concentration was measured using either NanoDrop 8000 Spectrophotometer (Thermo Fisher Scientific) or the Qubit Fluorometer (Thermo Fisher Scientific).

Cells were seeded at 1 × 10⁶ cells per 10-cm culture dish and cultured overnight. MCF10A-ER-Src cells were treated by 1 µM 4-hydroxy-tamoxifen for various time points (1, 4, and 24 hr) to induce transformation. Cells were pretreated with cycloheximide (100 µg/ml; Sigma-Aldrich, St. Louis, MO) for 90 s or harringtonine (2 µg/ml; Santa Cruz, Santa Cruz, CA) for 5 min, and detergent lysis was then performed with flash-freezing in liquid nitrogen. For ribosome profiling, DNase I-treated lysates were then treated with RNase I, and ribosome-protected fragments were purified for Illumina TruSeq library construction as previously described (Ingolia et al., 2012 (link)). For RNA-seq, total RNA was purified from DNase-treated lysates, and ribosomal RNA was depleted with RiboMinus Eukaryote Kit (Thermo Fisher Scientific, Waltham, MA). RNA-seq libraries were prepared with a tagging-based workflow (Pease and Kinross, 2013 ). In brief, rRNA-depleted RNA was fragmented at 85°C for 5 min, followed by cDNA synthesis, terminal tagging and PCR amplification with ScriptSeq v2 RNA-Seq Library Preparation Kit (Epicentre, Madison, WI). Ribosome profiling and RNA-seq libraries were sequenced with Illumina HiSeq 2500.