Computational protocol: Comprehensive transcriptomic and proteomic characterization of human mesenchymal stem cells reveals source specific cellular markers

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[…] Total RNA was extracted with TRIZOL and further cleaned on RNAeasy columns (Qiagen). RNA quality was evaluated by the Agilent Bioanalyzer 2100 system. Only preparations with RIN (RNA integrity number) values above 8 were considered.Following RNA isolation, 100 ng of total RNA was converted to cDNA using the Ovation RNA-seq System V2 (Nugen Technologies, San Carlos, CA). 2 μg of the amplified cDNA was sheared to 150–200 bp size distribution by Adaptive Focused Acoustics using a Covaris E220 instrument (Covaris, Woburn, MA) under the following conditions: 50 μl total volume, 20% duty cycle, 175 intensity, 200 cycles per burst, 165 sec in frequency sweeping mode. The remainder of the library preparation followed manufacturer’s protocol as described in Encore Rapid IL Multiplex System (Nugen Technologies). Briefly, the sheared cDNA was end-repaired to generate blunt ends then ligated to Illumina compatible adaptors with indexing tags, followed by 1× AMPure XP beads purification. The final NGS libraries were quantified using Agilent Bioanalyzer DNA Chip 1000 then pooled; 11 libraries per pool. Paired-end 100 bp deep sequencing was carried out on HiSeq 2500 (Illumina).Using a customized analysis pipeline () all samples were run together on a virtual machine. The analysis pipeline implemented as a bash shell script is composed of TopHat, Picard, (http://picard.sourceforge.net/index.shtml), Samtools and Cuffdiff. TopHat aligns RNA-seq reads to the reference genome (GRCh37). Picard removes PCR duplicates and Samtools creates indices for bam files. We ran the Cuffdiff function from the Cufflinks package v2.1.1. to find differentially expressed genes and transcripts in the samples using the default settings. Results from Cuffdiff were plotted in R using the Bioconductor CummeRbund package.Information for mapped, unmapped and duplicate reads was extracted using Samtools. The length of the transcript in base pairs was calculated by summing up depth per genome position. The covered fraction of transcript was computed by dividing the total number of bases in mapped reads with the length of the transcript. Depth per genome position was calculated using GenomeCov function from BedTools. RNA quality control was performed with RNA-SeQC. Read metrics are summarized in . […]

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