Trueseq stranded mrna platform

The qualified RNA (more than 100 μg and RIN > 7.0) was retained for sequencing in the next step. Then the poly (A) RNA solution was fragmented at 94 °C for 5 min using the thermocycler. Subsequently, the pre-equilibrated m⁶A-Dynabeads was added into the fragmented RNA at room temperature while rotating (tail-over-head) at 7 rotations per minute for one hour. After washing of m⁶A-Dynabeads, elution of m⁶A-positive RNA and extraction and cleanup step of the RIP, a RIP library was constructed using 100 ng of RNA (100 ng of input and 100 ng of post m⁶A-IP positive fraction) utilizing the Illumina TrueSeq Stranded mRNA platform. Finally, Illumina HiSeq X10 system (OE Biotech Co., Ltd., Shanghai, China) was performed to conduct the 2×150 bp paired-end sequencing. The raw data in this work was deposited at the GENE EXPRESSION OMNIBUS (GEO) database (accession number: PRJNA879097).

First, the quality and quantity of total RNA were estimated using Bioanalyzer 2,100 (Agilent, CA, United Stets) and NanoDrop 2000 (Thermo Scientific), both with an RIN number > 7.0. More than 100 μg of total RNA was used for mRNA isolation via an mRNA Purification Kit (Ambion Dynabeads mRNA Purification Kit). Subsequently, poly (A) mRNA fractions were created using divalent cations and a thermocycler (RNA solution kept at 94°C for exactly 5), incubated with m⁶A-Dynabeads (Anti-m⁶A, Synaptic Systems, Cat. No 202003) in an m⁶A-binding buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl₂, 1% NP-40, 2 mM EDTA), and allowed to bind to the beads. Following this, the m⁶A-Dynabeads were washed and m⁶A-positive RNA was eluted. The RIP was extracted and cleaned, and finally, m⁶A “enriched RNA” was collected. We used 100 ng of RNA (100 ng of input and 100 ng of post m⁶A-IP positive fraction) for library construction with the Illumina TrueSeq Stranded mRNA platform. Finally, paired-end sequencing was performed on an Illumina HiSeq X10 system at OE Biotech Co., Ltd. (Shanghai, China) using manufacturer’s instructions. The data was submitted to the GENE EXPRESSION OMNIBUS (GEO) database (accession number GSE181266).

Sequencing libraries (Supplementary material) were prepared with the Illumina TrueSeq Stranded mRNA platform as in prior reports (34 (link)). The Illumina HiSeq X10 System at OE Biotech Co., Ltd. (Shanghai, China) was used to perform paired-end sequencing. All data were submitted to the Gene Expression Omnibus (GEO) database (Accession number: GSE205649). RNA-seq data were subjected to quality control and statistical analyses (Supplementary material) as in prior reports (34 (link)).