Mild and ultrafast GLORI enables absolute quantification of m6A methylome from low-input samples

HEK293T cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37C; Male C57BL/6J mice (6-week-old) were housed in a specific-pathogen-free barrier. The barrier maintained 12-hour light-dark cycle and 22 ± 1°C room temperature with ~50% humidity. mRNA was purified by oligo (dT) beads. Mice hippocampus samples were performed Ribo-off treatment. RNA were performed for GLORI 2.0/3.0. RNA libraries were prepared using SMARTer Stranded Total RNA-Seq kits (v2) - Pico Input mammalian (Takara) Library Prep Kit following manufacturer's protocols. Raw reads were treated by Trim Galore (v0.6.6) for adaptor trimming and quality control with the following parameters: "--nextseq 20 --phred33 --clip_R1 3 --stringency 1 -e 0.3 --length 25". Then trimmed reads were deduplicated by Seqkit (v0.13.2) using the following parameters: "seqkit rmdup -j 20 -s". Clean reads were mapped to A-to-G converted versions of the genome with STAR (v2.7.5c) and then transcriptome with bowtie (v1.3.0). The high-confidence m6A sites were identified and quantified by GLORI-tools (https://github.com/liucongcas/GLORI-tools).