Computational protocol: N6-methyladenosine alters RNA structure to regulate binding of a low-complexity protein

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[…] RNA-seq experiments were performed on two replicates of RNA samples from HNRNPG knockdown (KD1 and KD2) as well as control HEK293T cells 48 h after transfection. Total RNA was extracted using the RNeasy Plus kit (74104, Qiagen). Libraries were prepared using the TruSeq Stranded mRNA LT Sample Prep Kit (RS-122-9005DOC, Illumina). KD and control samples were sequenced together in four lanes in one flow cell. All samples were sequenced by Illumina HiSeq 2000 with paired-end 100-bp reads. The reads from the four lanes of each sample were combined for all analyses. The RNA-seq data were mapped using the splice-aware alignment algorithm TopHat version 1.1.4 () based on the following parameters: tophat –num-threads 8 –mate-inner-dist 200 –solexa-quals –min-isoform-fraction 0 –coverage-search-segment-mismatches 1. Gene expression level changes were analyzed using Cuffdiff (). Approximately 140–200 million reads were mapped for each sample. Differential splicing was determined using DEXSeq () based on Cufflinks-predicted, non-overlapping exons. [...] Phylogenetic conservation analysis was performed by comparing PhyloP scores at high-confidence m6A-containing AGRAC motifs to those at randomly selected AGRAC sequences. The PhyloP scores were accessed from the precompiled PhyloP scores () (ftp://hgdownload.soe.ucsc.edu/goldenPath/hg19/phyloP46way/) for primates and vertebrates. P-values were evaluated using the Mann–Whitney–Wilcoxon test. The random selection was done separately for primates and for vertebrates.Sequence logos were generated using the WebLogo package. R statistical package was used for all statistical analysis (unless stated otherwise). […]

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