Rna fragmentation buffer

The procedure was adapted from the previous report^{8 (link)}. For m¹A-qRT-PCR, total RNAs were firstly fragmented to 150 nucleotide using RNA fragmentation buffer (Ambion, AM8740), and then fragmented RNA (as input) was denatured and incubated with m¹A antibody for 2 h, and protein A/G magnetic beads were added to the mixture and incubated for additional 2 h. Beads were washed with buffer and purified, input and immunoprecipitated RNAs were reversed transcribed into cDNA using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific), and then quantified by qPCR using SYBR GREEN mix (TOYOBO). For comparing m¹A abundance changes, relative enrichment was first normalized with inputs, and then analyzed by comparing the data from m¹A-immunoprecipitated sample. All samples were analyzed in triplicate qPCR.

For transcriptome analysis purposes: cDNA preparation and Illumina HiSeq^TM sequencing was performed at the Beijing Genome Institute (BGI) as described by Ren et al.^{56 (link)}. The cDNA libraries were prepared according to the manufacturer’s instructions (Illumina, San Diego, CA). Beads coated with oligo(dT) were used to isolate eukaryotic poly(A) mRNA from the total RNA. Purified mRNA was then fragmented in RNA fragmentation buffer (Ambion, Austin, TX). Using these short fragments as templates, random hexamer-primers (Illumina) were used to synthesize the first-strand cDNA. The second-strand cDNA was synthesized using buffer, dNTPs, RNase H and DNA polymerase I. Short double-stranded cDNA fragments were purified with a QIAquick PCR extraction kit (Qiagen) and eluted with elution buffer for end repair and the addition of a terminal ‘A’ nucleotide. Next, Illumina sequencing adaptors were ligated to the DNA fragments. DNA fragments of a selected size were gel-purified and amplified by PCR. The amplified library was sequenced on an Illumina HiSeq^TM 2000 sequencing machine at BGI, using single-end sequencing with an expected library size of 160 bp and a read length of 90 nt.
For RT-qPCR purposes: The cDNA synthesis was preformed using the RevertAid RT kit (Thermo Scientific, Waltham, MA) in four times 40 µl volumes according to the manufacturer’s instructions.

In the present study, we used our WTTS-Seq protocol^{23 (link),81 (link)} to construct libraries for all rats. For each individual sample, 2.5 μg of total RNA was chemically fragmented using RNA fragmentation buffer (AM8740, Ambion) as the first step, then enrichment of poly(A) + RNA was accomplished with Dynabeads oligo(T) magnetic beads (61002, Ambion) followed by reverse transcription with SuperScript III Reverse Transcriptase (18080, Invitrogen) to synthesize first strand cDNA. Next, both 5′- and 3′- adaptors were added to fit the Ion Torrent sequencing platform. All RNA molecules were removed by both RNases H (M0297L, NEB) and I (EN0601, Thermo Scientific) to prevent contamination, 250–500 bp first-strand cDNA molecules were selected with solid-phase reversible immobilization beads (A63880, Beckman Coulter), and second-strand cDNA was synthesized by PCR. Lastly, all libraries were sequenced using an Ion PGM™ Sequencer at Washington State University.