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Pipeline publication

[…] or miRNA sequencing, the resulting pooled libraries were hybridized to oligonucleotide-coated single-read flow cells for cluster generation using HiSeq® Rapid SR Cluster Kit v2 on Hiseq 2500 (Illumina, USA). The clustered pooled miRNA libraries were then sequenced on the HiSeq 2500 for 50 sequencing cycles using HiSeq® Rapid SBS Kit v2 (50 Cycle) (Illumina, USA). As for mRNA sequencing, each library were subjected to clonal amplification on Ion Chef System using Ion PI IC 200 kit followed by sequencing using PI BC v2 chip on Ion Proton system (all from Life Technologies, USA). Two to three mRNA libraries were pooled into a single chip., Preprocessing of miRNA sequencing data was executed in BaseSpace software (Illumina, USA), and FASTQ files were generated. MiRNA Analysis app version 1.0.0 was used for determination of differentially expressed (DE) miRNAs using the workflow described by Cordero et al. (). Briefly, the pipeline includes 3′ end adapter removal using cutadapt (), annotation to miRBase v21 (), mapping using SHRIMP aligner (), and differential analysis of miRNAs using DESeq2 (). In addition, Rank Product statistics were used to detect 3p/5p ratio changes on the same miRNA in two different conditions (). Unsupervised hierarchical clustering and heatmaps were performed and created using Morpheus from Broad Institute (https://software.broadinstitute.org/morpheus/).Pathway enrichment analysis were executed using DIANA-miRPath v3.0 () and significance was determined using Fisher’s Exact Test., For mRNA sequencing, preprocessing of data were conducted on Torrent Server using Torrent Suite v4.4.2 software (Life Technologies, USA) and FASTQ files were generated and then exported for data analysis using CLCBio Genomics Workbench v8.3 (CLC Bio, Denmark). The manufa […]

Pipeline specifications

Software tools BaseSpace, cutadapt, DESeq2
Databases miRBase