GSM6004978: shSCR_rep_2; Homo sapiens; RNA-Seq
RNA was extracted using the RNeasy Mini Kit with on-column DNase (Qiagen) and further purified with the RNA Clean and Concentrator Kit (Zymo Research). Samples were quality-checked using the Agilent RNA 6000 Nano Kit and Agilent 2100 BioAnalyzer (Santa Clara, CA); all samples had RNA integrity values of >8.5. Poly(A) mRNA enrichment and library preparation were performed using the NEBNext Poly(A) mRNA Magnetic Isolation Module, NEBNext Ultra II RNA Library Prep Kit (New England BioLabs) with SPRIselect Beads (Beckman Coulter), and NEBNext Multiplex Oligos for Illumina (index primers sets 1 and 2) according to the manufacturer's instructions. Library sizes were checked with the Agilent DNA 1000 Kit, and library concentrations were determined with Qubit dsDNA assays (Thermo Fisher Scientific). Equimolar amounts of each library were pooled, and pools were diluted to a final concentration of 1.8 pM in HT1 Hybridization Buffer (Illumina) and sequenced on an Illumina NextSeq 500 instrument using the NextSeq 500/550 75-cycle High-Output Kit v2.5.