Computational protocol: A compendium of long non-coding RNAs transcriptional fingerprint in multiple myeloma

Similar protocols

Protocol publication

[…] Total RNA was extracted from purified PCs by using Trizol reagent. Quantitative assessment of the RNA was performed using Nanodrop ND-1000 Biophotometer (NanoDrop Technologies): the minimum OD 260/280 ratio to be considered acceptable is 1.98–2.10. Four-hundred ng of total RNA were used to prepare paired-end (PE) cDNA libraries using the TruSeq® RNA Sample Preparation kit for total RNA (Illumina). The libraries were sequenced to obtain strand-specific 100 bp PE reads on a HiSeq. 2000 (Illumina). Reads were aligned to the human genome using STAR under default conditions and Gencode v25 GTF file. STAR aligner was based on splice junctions from the Ensembl database version 87. Transcript abundance was estimated using featureCounts (default parameters). FPKM (Fragments Per Kilobase Million) quantification was performed on sorted BAM files using cufflinks default procedure. Differentially expressed genes were identified using DeSeq at FDR < 0.01, provided that expression across the whole dataset was not null. Quality Control (QC) analysis was performed using multiqc tool and the QC metrics were comparable for all samples. The annotation allowed to detect 14,202 lncRNAs, including the following Gencode biotypes: lincRNA, antisense, bidirectional promoter lncRNA, sense intronic, sense overlapping, 3′ overlapping ncRNA. The expression filter retained 9,540 lncRNAs in our dataset. […]

Pipeline specifications

Software tools Subread, Cufflinks, DESeq, MultiQC
Databases GENCODE
Application RNA-seq analysis
Organisms Homo sapiens
Diseases Multiple Myeloma, Neoplasms