Truseq stranded total rna kit

RNA was extracted from TRIzol-lysed PBMCs using the miRNAeasy Mini Kit (Qiagen). RNA quantity was measured on the Nano Drop 2000 Spectrophotometer (Thermo Scientific) (56.6 ± 16.7 ng μl⁻¹) and the quality and integrity measured with the Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). All RNA integrity numbers (RINs) were greater than 6 (RIN: 7.66 ± 0.79). The Illumina TruSeq Stranded Total RNA kit (Ilumina, San Diego, CA, USA) was used for library preparation accordingly to manufacturer instructions without any modifications. A total of 80 indexed RNA libraries were pooled and sequenced using long read paired-end chemistry (2 × 150 bp) at a read depth of 30 M reads per sample using the Illumina HiSeq2500. Adapter sequences were clipped and low quality reads were discarded using Trimmomatic^{27 (link)} using parameters: ILLUMINACLIP, MINLEN:140, CROP:140. All high-quality trimmed reads were then mapped to UCSC Homo sapiens reference genome (build hg37) using default STAR v2.4.0 parameters²⁸ (percentage of mapped reads: 92.1% ± 1.6%). Samtools was used to convert bamfiles to samfiles and featureCounts^{29 (link)} was used to quantify gene expression levels for each individual sample using default paired-end parameters.

The amount of total RNA used for sequencing was 1 µg. To maximize mRNA representation in the metatranscriptomic libraries, the Ribo-Zero rRNA removal kit (Bacteria) (Illumina, San Diego, CA, USA) was used to remove rRNAs from the total RNA preparations. Library preparation was performed using the Illumina TruSeq stranded total RNA kit (Illumina). Sequencing was carried out using an Illumina HiSeq2000 platform. In total, 178 million of paired-end reads were generated from nine independent libraries: three libraries from rumen solid samples, three libraries from cardoon biomass digested for 48 h, and three libraries from cardoon biomass digested for 96 h.

We extracted total RNA from 20 pairs of ovaries of females aged 3–5 days per replicate per genotype. For each genotype (HOAP[mel] and HOAP[yak]), we prepared three biological replicates for a total of six samples. We dissected ovaries into cold 1XPBS and proceeded with RNA extraction using the standard Trizol-based protocol (Invitrogen, Carlsbad, CA). To remove DNA contamination, we used TURBO DNase (Thermo Fisher Scientific, Waltham, MA) then purified samples using a Qiagen RNeasy kit (Qiagen, Hilden, DE). For total mRNA sequencing, the Weill Cornell Epigenetics Core performed ribosomal RNA depletion using Ribo-Zero depletion kit (Illumina, San Diego, CA) and prepared libraries using the Illumina TruSeq Stranded Total RNA kit (Illumina, San Diego, CA). The Core sequenced 200 ng per sample on a HiSeq 2500 using SBS kit v4 on a single-end flow cell (50 cycles). For small RNA sequencing, Fasteris SA (Geneva, CH) conducted acrylamide gel size selection and anti-2S treatment with proprietary oligos as well as standard library preparation protocols using an Illumina TruSeq small RNA kit. They sequenced the libraries on an Illumina NextSeq500 (run mode 1 × 50).

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)}.

RNA was isolated from four 20 μm sections of formalin-fixed paraffin-embedded (FFPE) tumor samples of the RA-QA cohort using the AllPrep DNA/RNA FFPE kit (Qiagen, Germany), followed by a quality control for purity and integrity by the Agilent Bioanalyzer system. Total RNA was depleted from ribosomal RNA and random primed for complementary DNA synthesis using the TruSeq-stranded total RNA kit (Illumina, USA). RNA-sequencing was performed on the Illumina HiSeq2500 platform (Illumina) with paired-end 25× coverage (PE100–125). The FASTQ files were trimmed to remove adaptor sequences using flexbar (v3.0.3, ref. ^{79 (link)}) and aligned to GRCh37/hg19 reference genome using hisat2 (v2.0.5, ref. ^{80 (link)}), resulting in an average 10–15 M aligned reads. Reads were counted to genomic features using subreads (v1.5.5, ref. ⁸¹ ). For both the TCGA and RA-QA cohort, RNA-seq data were corrected for GC content and normalized within and between lanes using the R package EDASeq (v2.12.0, ref. ^{82 (link)}), and quantile normalized using the preprocessCore (v1.36.0, ref. ⁸³ ).