GSM8666311: LNCaP_Control; Homo sapiens; RNA-Seq
RNA Extraction: RNA extraction was performed using the Trizol-chloroform method as described previously 15. The total RNA concentration was calculated by Quant-IT RiboGreen (Invitrogen, cat#R11490). To assess the integrity of the total RNA, samples are run on the TapeStation RNA ScreenTape (Agilent, cat#5067-5576). Only high-quality RNA preparations with RIN greater than 7.0 were used for RNA library construction. A library was independently prepared with 1μg of total RNA for each sample by Illumina TruSeq Stranded mRNA Sample Prep Kit (Illumina, Inc., San Diego, CA, USA, cat#20020595). The poly-A-containing mRNA molecules were purified using poly‐T‐attached magnetic beads, followed by mRNA fragmentation into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were copied into first-strand cDNA using SuperScript II reverse transcriptase (Invitrogen, cat#18064014) and random primers, and subsequently used for second-strand cDNA synthesis using DNA Polymerase I, RNase H, and dUTP. These cDNA fragments underwent an end-repair process by adding a single adenine (A) base and ligating the adapters. The products were purified and enriched with PCR to create the final cDNA library. The libraries were quantified using KAPA Library Quantification kits for Illumina Sequencing platforms according to the qPCR Quantification Protocol Guide (KAPA BIOSYSTEMS, #KK4854) and qualified using the TapeStation D1000 ScreenTape (Agilent Technologies, # 5067-5582). The indexed libraries were then submitted to an Illumina NovaSeq (Illumina, Inc., San Diego, CA, USA), and the paired-end (2×100 bp) sequencing, which was performed by Macrogen Inc., Korea. The experiments were run in duplicate, but cells were pooled at the library preparation step.