m6A-seq Analysis of Murine Dendritic Cells

Total RNA was isolated from DCs. Polyadenylated RNA was further enriched from total RNA by using Dynabeads® mRNA Purification Kit (Invitrogen). RNA samples were fragmented into ~100-nucleotide-long fragments with sonication. Fragmented RNA (100 ng mRNA or 5 μg total RNA) was performed m⁶A-IP following EpiMark N⁶-methyladenosine enrichment kit (NEB E1610S) protocol. RNA was enriched through RNA Clean&Concentration-5 (Zymo Research) and used for library generation with SMARTer Stranded Total RNA-Seq Kit (Takara). Sequencing was performed at the University of Chicago Genomics Facility on an Illumina HiSeq4000 machine in single-read mode with 50 bp per read. Sequencing reads were aligned to the mouse genome mm9 by STAR (version 2.6.0c)^{29 (link)}. The m⁶A-enriched regions (peaks) in each m⁶A-IP sample were detected by MACS2 (version 2.1.1.20160309)^{30 (link)} with q value less than 0.01 and corresponding m⁶A-Input sample was used as the control. Peaks that were detected by both replicates were considered as high confident peaks. The peaks annotation and binding motif were analyzed by HOMER (version 4.9)^{31 (link)}.