Computational protocol: RNA-Seq following PCR-based sorting reveals rare cell transcriptional signatures

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Protocol publication

[…] Positively sorted TaqMan emulsions were broken using perfluoro-1-octanol and the aqueous fraction was diluted in water. Total RNA was purified using the Quick-RNA Microprep kit (Zymo), performing on column DNA digestion with 5 μl DNAse I and 5 μl Exonuclease I (NEB) for 90 min at RT to decrease genomic and TaqMan PCR amplicon contamination in the downstream preparation steps. The RNA was recovered by performing two 8-μl elutions. After rRNA depletion (Ribo-Zero Gold kit, Illumina), libraries were prepared using the SMARTer Stranded RNA-Seq kit (Clontech) and amplified using 15 PCR cycles. The libraries were purified with Select-a-Size DNA Clean & Concentrator columns (Zymo) with a 75 bp cutoff, and eluted in 22 μl. Libraries were analyzed on a High Sensitivity DNA Assay chip with a Bioanalyzer (Agilent Technologies), and sequenced on a HiSeq4000 in single-end 50 bp multiplexed runs. Sequenced reads passing quality control (FastQC, cutadapt, trimmomatic, [, ]) were aligned to the hg19 human transcriptome (iGenomes) using TopHat2-Bowtie2 mappers [, ]. Downstream analyses were performed using samtools, HTSeq [], and the edgeR and gplots packages for R [, ]. The Benjamini–Hochberg procedure was used to control for multiple comparisons. […]

Pipeline specifications

Software tools FastQC, cutadapt, Trimmomatic, TopHat, Bowtie2, SAMtools, HTSeq, edgeR, gplots
Databases iGenomes
Application RNA-seq analysis
Organisms Homo sapiens
Diseases Prostatic Neoplasms