GSM6793068: N27_AML1_ENTI_2_4h_C; Homo sapiens; RNA-Seq
RNA was subjected to polyA selection using magnetic oligo-dT beads (Thermo Fisher Scientific, #61002). A 94 °C-incubation for 4 min partially fragmented polyA RNA. Both actinomycin-D (during the first-strand cDNA synthesis) and dUTP (during the second-strand synthesis) were used to keep the strand information. PolyA-enriched, strand-specific RNA libraries were generated with the TruSeq mRNA stranded sample preparation kit (Illumina, San Diego, CA, USA, #20020594) starting from 1 µg of RNA from each sample. RNA was subjected to polyA selection using magnetic oligo-dT beads (Thermo Fisher Scientific, #61002). A 94 °C-incubation for 4 min partially fragmented polyA RNA. Both actinomycin-D (during the first-strand cDNA synthesis) and dUTP (during the second-strand synthesis) were used to keep the strand information. The libraries were end-repaired and adenylated before being ligated with Y-shape single-indexed adapters and amplified by 11 PCR cycles. The last purification step was performed with Ampure XP beads (Beckman Coulter, #A63882) at 0.8x to remove all adapter dimers. Each library was quantified and quality-controlled using a Qubit 4 fluorometer (Thermo Fisher Scientific) and LabChip GX (Perkin Elmer, Waltham, MA, USA). The adaptor-tagged pool of libraries was loaded onto Illumina Hiseq2500 rapid-run flow cells (SR100 chemistry) for cluster generation and deep sequencing.