Ribo zero human mouse rat

Libraries were prepared starting with 300-500 ng of good quality total RNA (RIN ≥7.5) using the TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Human/Mouse/Rat (Illumina), according to the manufacturer's instructions. The kit contains 96 uniquely indexed adapter combinations in order to allow pooling of multiple samples prior to sequencing. After determining their size (with the Agilent 2100 Bioanalyzer and the Agilent High Sensitivity DNA Kit by Agilent Technologies) and concentration (by qPCR with the KAPA Illumina ABI Prism Library Quantification Kit, Kapa Biosystems), libraries have been pooled and sequenced (single-end, 125 bp) using a Single End V4 Cluster Kit and an Illumina HiSeq2500 or HiSeq4000 instrument.

Total RNA was extracted from purified mouse CD4⁺CD8⁺TCRβ^intCD24^high (thereafter referred to as DP) and CD4⁺CD8^-TCRβ^highCD24^low (hereafter referred to as CD4⁺ single-positive) cells using RNeasy Mini kit (Qiagen), including a DNaseI treatment step. RNA quality was determined using the 2100 Bioanalyzer instrument (Agilent) and RNA concentration was determined using Qbit 2.0 Fluorometer (Life Technologies). To examine expression levels and splicing patterns in mouse cells, 300 ng of total RNAs per sample (three biological replicates each) were used for library preparation using the TruSeq Stranded Total RNA kit with Ribo-Zero Human/Mouse/Rat (RS-122-2203, Illumina). The libraries were sequenced on an Illumina HiSeq 2500 instrument in 75 bp paired-end mode using 0.5 lanes per sample.

RNA was extracted from paired diagnosis/relapse samples of five pts with NPM1^mut loss and five pts with NPM1^mut persistence using the AllPrep DNA/RNA Mini Kit (QIAGEN), and RNA quality was assessed using a BioAnalyzer 2100 (Agilent). Libraries were prepared from 1 µg of total RNA using the TruSeq Stranded Total RNA Kit with Ribo-Zero Human/Mouse/Rat (Illumina) according to the manufacturer’s instructions. The pooled RNA libraries were sequenced on an Illumina HiSeq2000 to obtain 100 bp paired-end reads. RNA-Seq reads were aligned to the human reference genome hg 19 and quantified using STAR v.2.4.2a⁵⁶ in the 2-pass mapping mode. Furthermore, the DESeq2^{57 (link)} package from R was used to obtain normalized expression values. BRB-ArrayTools Version 4.5.0 Beta_2^{58 (link)} (BRB, National Cancer Institute, Bethesda, MD, USA) and GSEA (http://broadinstitute.org/gsea/index.jsp)^{51 (link)} were used for class comparison analyses. Hierarchical clustering and heatmap visualization of differentially expressed genes was performed using Cluster 3.0^{59 (link)} and Java Treeview^{60 (link)}.

For library preparation, 1 μg of total RNA and the TruSeq Stranded Total RNA Library Prep kit® with Ribo‐Zero Human/Mouse/Rat (Illumina, Inc., San Diego, CA, USA) were used. Fragmentation step was omitted in most of the samples due to sample quality, while in five samples, it was done according to recommendations from Illumina®. Validation of libraries was performed in the Agilent 2100 Bioanalyzer® system and then normalization to 10 nM was done with the Qubit dsDNA HS Assay kit® (ThermoFisher Scientific, Wilmington, USA), before cluster generation. Sequencing was performed in 12‐pooled samples at 1 × 75 bp with single‐end strategy in a NextSeq 500® (Illumina Inc., San Diego, CA, USA), with no sequencing results in six tumor samples.