Allprep dna rna column based extraction kits

RNA was extracted from FACS-sorted cells using Qiagen Allprep DNA/RNA column-based extraction kits as per the manufacturer’s instructions. RNA-seq was performed on DNase-treated samples using an RNA-seq lite plate-based protocol with SMART cDNA amplification. Non-aberrant cell subsets with the highest RNA quality for each subject were selected for RNA-seq library construction and sequencing (> 10 ng RNA and RNA quality score ≥ 6.4), as these were most likely to produce informative sequencing data to identify a dominant clone among immunophenotypically normal T cells. All samples were initially subjected to shallow sequencing (average 4.1 million total reads per sample, range 2.4–6.0 million) to facilitate the identification of the malignant T-cell clone in the sorted populations. To evaluate the importance of sequence depth for this application, all samples were re-sequenced deeply (average 92 million total reads per sample, range 63–156 million). All sequencing was performed using 125 nucleotide paired-end reads on an Illumina HiSeq 2500 instrument at the Genome Sciences Centre in Vancouver, Canada.