GSM8488531: BC4; Homo sapiens; RNA-Seq
Total RNA was extracted from FFPE slides using QIAGEN RNeasy FFPE kit (standard kit) following Manufacturer's instructions (Qiagen, Hilden, Germany). RNA samples were quantified using Qubit 2.0 Fluorometer RNA assay (Invitrogen, Carlsbad, CA, USA) and RNA integrity was checked with Agilent 4200 TapeStation (Agilent Technologies, Palo Alto, CA, USA). RNA library preparations, sequencing reactions, and initial sequencing data quality analysis were conducted at GENEWIZ, LLC. (South Plainfield, NJ, USA). RNA sequencing library preparation was prepared using TruSeq Stranded Total RNA library Prep kit following manufacturer's protocol (Illumina, Cat#20020597). Briefly, rRNA was depleted and fragmented for 8 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized. The second strand of cDNA was marked by incorporating dUTP during the synthesis. cDNA fragments were adenylated at 3'ends, and indexed adapter was ligated to cDNA fragments. Limited cycle PCR was used for library enrichment. The incorporated dUTP in second strand cDNA quenched the amplification of second strand, which helped to preserve the strand specificity. Sequencing libraries were validated using DNA Analysis Screen Tape on the Agilent 2200 TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (Applied Biosystems, Carlsbad, CA, USA).