Computational protocol: Polypyrimidine tract binding protein 1 protects mRNAs from recognition by the nonsense mediated mRNA decay pathway

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Protocol publication

[…] For RNA-seq, 1 μg of each total RNA sample was used for library preparation. Ribosomal RNAs (rRNAs) were subtracted from total RNA with Ribo-Zero rRNA Removal Kits (Human/Mouse/Rat; Epicentre). Following purification, RNA was fragmented and converted into first strand cDNA using reverse transcriptase and random primers, followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. Following adapter ligation, products were purified and amplified by PCR to create final cDNA libraries. cDNA libraries were validated using an Illumina Miseq and paired-end 50 bp sequencing was performed on an Illumina HiSeq 2000 instrument. Raw data in FASTQ format was aligned to the reference genome hg19 using TopHat2/bowtie2. Reads mapping to the 3’UTRs of RefSeq genes were counted by Bedtools. Every RefSeq transcript and its 3’UTR expression level were normalized by three steps: calculating raw reads into RPKM (reads per-kilobase-per million), 75th percentile normalization and log2 transformation. The dataset was filtered to remove mRNAs represented by fewer than 25 3’UTR-mapped reads, and differentially expressed genes were selected by one-way ANOVA analysis (; p<0.05). The RefSeq transcript with the longest annotated 3’UTR for each gene was used for construction of the reference set of mRNAs used in all subsequent analyses (designated 'exemplar' RefSeq mRNAs). For , the six most highly enriched hexamers in PTBP1 CLIP experiments (; UUCUCU, UCUUCU, UCUCUU, UCUCUG, CUUUCU, and CUUCUC), representing over 50% of all PTBP1 CLIP peaks, were used. […]

Pipeline specifications

Software tools TopHat, Bowtie2, BEDTools
Organisms Homo sapiens
Diseases Dental Caries