GSM5202132: Patient-derived GSC -BT13 shCTRL D6-; Homo sapiens; RNA-Seq
Total RNA was extracted from lysed cells or gliospheres using the RNeasy Mini kit (#74104) (Qiagen) according to the manufacturer's instructions. After DNase treatment, the quality of the RNA preparations (1 µg) was controlled using a Tapestation (Agilent). Total RNA 1 ug is treated with Illumina's Ribo-Zero Complete Gold Kit to remove the ribosomal RNA. First the samples are treated with rRNA removal solution after which the rRNA is removed combining probe-hybridized samples and magnetic beads. The Ribo-Zero treated RNA is purified with Qiagen RNeasy MinElute Cleanup Kit. The absence of rRNA is assessed by Bioanalyzer and the quantity of RNA by Bioanalyzer. NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB #E7420S/L Version 8.0 3/16) is used to generate cDNA libraries for next generation sequencing. First, the ribosomal RNA depleted sample (10 ng) is fragmented to generate inserts around 200 bp, and then primed with random primers. The first strand cDNA synthesis utilizes Actinomycin D, which inhibits the DNA polymerase activity of the reverse transcriptase increasing strand specificity. In the second strand cDNA synthesis dUTP labelled oligo nucleotides are incorporated to mark the second strand with uracils (U). The cDNA synthesis product is purified with Agencourt AMPure XP beads. Next, the cDNA is end-repaired, and adapter ligated utilizing dA-tailing. The adaptor ligated DNA goes through a bead-based size selection after which the final PCR enrichment takes place. At this point, each sample is given a unique index to enable pooling of multiple samples (multiplexing) for sequencing. During the high-fidelity PCR, USER (Uracil-specific Excision Reagent) enzyme cuts away the uracil strand preserving only the first strand. In addition, the loop adaptor is cut open to enable the PCR. The amplified library is then purified using AMPure XP Beads. Library quality is assessed by Bioanalyzer (Agilent DNA High Sensitivity chip) and library quantity by Qubit (Invitrogen). Illumina NextSeq 500 High Output 75 cycle kit for single-end 1x75bp reads (2 flow cells). Loading concentration 1,5 pM + 1% PhiX control. Starting amount of Total RNA: 1000 ng, PCR cycles used in method: 15.