GSM5292566: TS153_36_RNA_603_SOX4_KD3-2_6D; Homo sapiens; RNA-Seq
Total RNA was extracted from 5x105  AML patient leukemic blasts using QIAshredder spin columns and a RNeasy Plus Micro kit (QIAGEN). Extracted RNA was DNAse treated using the TURBO DNA-free Kit (Ambion, Austin, TX) according to the manufacturer's instructions. Before sequencing, RNA integrity was checked using a 2100 Bioanalyzer (Agilent Technologies). RNA-seq libraries were prepared using either the Truseq Stranded Total RNA kit (Illumina, San Diego, CA) or Lexogen Quantseq 3' (Lexogen, Vienna, Austria) according to the manufacturer's instructions. The Truseq kit includes a ribo-depletion step to eliminate ribosomal RNAs and it captures coding RNA plus multiple forms of non-coding RNA while the Quantseq is designed for 3' end sequencing of polyA RNA. Libraries were quantified by quantitative PCR using a Kapa Library Quantification Kit for Illumina sequencing platforms (Roche). Libraries were then barcoded, pooled and sequenced using either a Novaseq6000 or a NextSeq500 desktop sequencing system (Illumina). A single run (400 million reads) of 75 bp paired-end sequencing produced a mean of 49.4-54.72 million reads per sample in the case of Truseq libraries or 100bp single-end with a mean of 17 million reads per sample in the case of Quantseq 3' libraries.