GSM7770418: RPE p53-/-, parental control, replicate 3; Homo sapiens; RNA-Seq
RNA samples were prepared using RNeasy Mini Kit and RNase-Free DNase Set (QIAGEN). RNA samples were quantified using Qubit 2.0 Fluorometer, and the RNA integrity was checked with 4200 TapeStation. NEBNext Ultra II RNA Library Prep Kit for Illumina was used following the manufacturer's recommendations (New England BioLabs). Briefly, mRNAs were initially enriched with Oligo d(T) beads. Enriched mRNAs were fragmented for 15 minutes at 94C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3´ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. The sequencing libraries were validated using Agilent TapeStation and quantified using Qubit 2.0 Fluorometer as well as by quantitative PCR (KAPA Biosystems). The sequencing libraries were clustered on flowcell lanes. After clustering, the flowcell was loaded on the Illumina NovaSeq instrument according to manufacturer's instructions.