Computational protocol: m6A mRNA methylation controls T cell homeostasis by targeting IL-7/STAT5/SOCS pathway

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Protocol publication

[…] We performed the Ribosome profiling by strictly following the manual of Illumina TruSeq Ribo Profile (Mammalian) Kit. Briefly, 50 million of Naïve T cells were isolated from WT and Mettl3 KO mice, and washed and treated with 0.1 mg/ml cycloheximide for 1min. After lysis, 1/10 of the cell lysate were used to isolate RNA for preparing input RNA library, the remaining were used to prepare ribosome footprints by first digested with 60U of 10 U/ul nuclease, then cleaned up by MicroSpin S-400 columns. The total RNAs and ribosome protected RNAs were then isolated with RNA Clean & Concentrator-25 kit (Zymo Reseaarch), and subjected to rRNA removal with Illumina Ribo-Zero Gold Kits. Polyacrylamide gel electrophoresis (PAGE) was used to purify the 28nt and 30nt long ribosome protected fragments, which were ligated 3′ adapters and prepared cDNA library. The cDNA library were PAGE gel purified again for the 70–80nt fragments, circularized, and amplified by PCR for 9 cycles, the resulting libraries were cleaned up by AMPure XP beads, purified by PAGE purification for 140~160bp fragments, and subjected to Illumina Hi-Seq for 75bp paired-end sequencing.We used the tuxedo suite (https://ccb.jhu.edu/software/tophat/) to align the ribosome profiling reads and determine FPKM, fold change, and the associated p-value. All genes which did not have enough reads aligned to determine FPKM were discarded, and the remaining genes were plotted. To calculate translation efficiency, HTseq was used to count the number of reads in each experiment. The normalized translation efficiency was calculated by taking the log2 of the quotient of the ribosome footprint read count divided by the mRNA-Seq read count. The individual graphs were plotted using R package (https://bioconductor.org/packages/release/bioc/html/Sushi.html). […]

Pipeline specifications

Software tools Tuxedo, TopHat, HTSeq, Sushi.R
Application Ribo-seq analysis