Rna fragmentation reagent

A C. hainanensis leaf transcriptome library was constructed using an mRNA-seq assay for paired-end Illumina sequencing, which was performed at Majorbio Biopharm Technology Co., Ltd. (Shanghai, China). Poly(A) mRNA was isolated from total RNA by using Sera-mag Magnetic Oligo (dT) Beads (Thermo Fisher Scientific, USA), and then mRNA-enriched RNAs were chemically fragmented to short pieces using the RNA Fragmentation Reagent (Ambion, USA). Double-stranded cDNA was synthesized using the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen, Carlsbad, CA). Subsequently, the Illumina Paired End Sample Prep kit (Illumina, USA) was used to construct a RNA-seq library which then was sequenced by Illumina HiSeq 2000 (Illumina, San Diego, CA).

m6A profiling of glioma stem samples before and after miRNA overexpression was performed using the Magna MeRIP m6A Kit according to manufacturer instructions. mRNA was isolated from glioma stem and differentiated samples using the Dynabeads mRNA Purification Kit (Thermofisher) and subsequently fragmented with RNA Fragmentation Reagent (Ambion) prior to immunoprecipitation. Total RNA was isolated from identical samples using Trizol (Ambion) and fragmented in the same manner. DNA libraries were then prepared using a custom Qiaseq Targeted RNA panel (Qiagen, CRHS-10308Z-88). cDNA from each RNA sample was assigned molecular barcodes followed by a 2-step PCR amplification with intermittent cleanup between each step via QIAseq beads as described by the manufacturer. PCR products were then quantified and sequenced using MiSeq (Illumina).

For the preparation of each strand-specific RNA library we used between ~50 ng (ring stage parasites) and ~150 ng (trophozoites) mRNA. mRNA was fragmented to approximately 100-200 nt in length. Reproducible fragmentation was obtained by mixing RNA with a RNA fragmentation reagent (Ambion) and heating it to 70°C for exactly 5 min. To remove the 5′-terminal 7-methylguanylate cap, the fragmented RNA was treated with 10U of Tobacco Acid Pyrophosphatase (Epicentre) for 2 hours at 37°C. All subsequent steps were performed according to an Illumina application Note (Note: directional mRNA-Seq sample preparation) with the following exceptions: Custom-made 5′- adapter (5′-GUUCAGAGUUCUACAGUCCGACGAUCNNNN-3′, conc. 20 μM) was used instead of the RNA adapter provided by Illumina, the PCR amplification was performed for 12-16 cycles using the KAPA HiFi DNA polymerase (Kapabiosystems) and KAPA HiFi Fidelity Buffer according the manufacturer’s instructions (Mg² concentration was adjusted to 2.5 mM). The PCR product was purified and concentrated using AMPure XP beads (Beckman Coulter). Quality and concentration of all libraries was determined using a Bioanalyzer 2100 (Agilent) and high throughput sequencing was performed on a HiSeq2000 (Illumina) except for library 11, Figure 
2, which was sequenced on a Genome Analyzer (Illumina). For read statics see Figure 
2.

For RNAseq library construction, rRNAs were removed from total RNAs with the RiboMinus Plant kit (ThermoFisher, Waltham, MA, USA) for mRNA enrichment. The mRNAs were degraded into 300-nt fragments with an RNA fragmentation reagent (Ambion, USA). cDNAs were synthesized by reverse transcription with random hexamer primers. RNAseq libraries were generated using an NEBNext Ultra RNA library prep kit (NEB, Ipswich, MA, USA), and 150-bp pair-end RNA sequencing was conducted using the Illumina NovaSeq 6000 platform at the Personalbio company. Reads of RNAseq data were mapped to the rice genome v7 and the PXO99A genome separately [54 (link)]. The significance of differentially expressed genes (DEGs) was determined by a fold change ≥ 2 and a p-value < 0.05.

Total RNA was isolated from glioma stem and differentiated samples using Trizol and treated with RiboMinus (Thermofisher) to remove ribosomal RNA. Samples were then fragmented with RNA Fragmentation Reagent (Ambion) and 200ug of RNA was immunoprecipitated with m6A antibody (12ug) (Synaptic Systems, 202–003) and Dynabeads (Thermofisher) at 4°C overnight. The precipitated RNA was then used to perform RNA-seq using an Illumina-HiSeq2500 sequencer.