RNA-seq Library Preparation from FACS Cells

FACS isolated cells were lysed in Trizol (Ambion). RNA integrity was evaluated using a Eukaryotic RNA 6000 Nano chip on an Agilent 2100 Bioanalyzer (Agilent Technologies). Up to 1 μg of total RNA from each sample was used for library preparation with the TruSeq RNA Sample Preparation Kit (Illumina). A common adaptor was used for all samples and barcode sequences present in the reverse primer were introduced by 12–20 cycles of amplification as described (Pereira et al., 2013 (link)). Each library was assessed for quality and size distribution using an Agilent High Sensitivity Assay bioanalyzer chip and quantified by real-time PCR. Equimolar amounts of each barcoded library were mixed and single-end sequenced on an Illumina HiSeq Sequencing System.