m6A-modified RNA immunoprecipitation sequencing (MeRIP-seq) and analysis.

The quality of the RNA samples was assessed using an Agilent Bioanalyzer RNA 6000 Nano kit to ensure all RNA integrity numbers were >9.0. For poly(A) RNA purification, Dynabeads Oligo(dT)₂₅ (#61005, Life Technologies) were incubated with an aliquot of 75 µg RNA, washed, and subjected to a second incubation with the same RNA sample, according to the manufacturer’s instructions. RNA was obtained by ethanol precipitation overnight, fragmented using Ambion^® RNA Fragmentation Reagents (#AM8740, Life Technologies), and then repurified by ethanol precipitation overnight. An aliquot of fragmented RNA was retained as a control for RNA sequencing.

For MeRIP, fragmented RNA was denatured for 5 min at 75°C, cooled on ice for 2–3 min, and incubated with the anti-m⁶A-coupled beads in 300 µl of 140 mM IP Buffer for 2 h at 4°C. Unbound RNA was removed by centrifugation, and beads were washed three times with 140 mM IP buffer and then incubated in elution buffer (5 mM Tris HCl, pH 7.5, 1 mM EDTA, 0.05% SDS, and proteinase K) for 1.5 h at 50°C. The eluted RNA was extracted with phenol/chloroform and precipitated with ethanol. An aliquot of RNA was retained as a control for round 1 MeRIP RNA, and the remainder was subjected to a second round of MeRIP, as described above. Libraries for sequencing (input RNA-Seq and MeRIP-Seq) were prepared using Illumina TruSeq Stranded mRNA kits, entering the protocol at the Elute-Prime-Fragment step, with the modification that samples were heated to 80 °C for 2 mins to only prime but not further fragment. Samples were sequenced on a HiSeq 2000 across 2 lanes with single read 50. RNA-seq data were aligned to a combined hg19 (chr 1-22, X, Y) and HIV-1 (https://aidsreagent.org/, pLAI2) genome FASTA using the STAR aligner⁴⁶ keeping only uniquely mapping reads. Peaks were called with MeRIPPeR⁸, using Fisher’s Exact Test on 25 bp genome-based windows to test for statistically significant enrichment in the IP relative to the control input RNA, with Benjamini-Hochberg p-value adjustment and a p-value cutoff of 0.05. Windows were merged into peaks and peaks smaller than 100 bp were filtered out. Differentially methylated peaks were called by splitting the union of all peaks into 100 bp windows in 25 bp steps. MeRIP-seq and RNA-seq counts were tabulated using bedtools⁴⁷) and input into edgeR⁴⁸ to identify windows with significant changes in methylation. Read counts mapping to genes were obtained using HTSeq (HTSeq cite), gene features using R-Make count (http://physiology.med.cornell.edu/faculty/mason/lab/r-make/usage.html), and all other features using BedTools. RNA-Seq data are publicly available at GEO {"type":"entrez-geo","attrs":{"text":"GSE74016","term_id":"74016"}}GSE74016.

Gene ontology (GO) analysis of the uniquely methylated transcripts identified during HIV-1 infection was performed using AmiGO software. The top 10 categories are shown.