GSM8668767: PC-3AcT cells, replicate 2; Homo sapiens; RNA-Seq
Homogenizing Samples 1. Add 750 uL Trizol directly to the maximum of 1x10^7 cells. 2. Pipette this mixture tenderly. 3. Incubate for 5 minutes at room temperature for lysis.  Phase separation 1. Add 150 uL of chloroform per 750 uL of Trizol used for homogenization. 2. Shake tube vigorously by vortex for 15 seconds. 3. Incubate for 2–3 minutes at room temperature. 4. Centrifuge the sample at 12,000 × g for 15 minutes at 4°C. 5. Transfer the upper aqueous phase to a new tube. Total RNA concentration was calculated by Quant-IT RiboGreen (Invitrogen, #R11490). To assess the integrity of the total RNA, samples are run on the TapeStation RNA screentape (Agilent, #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 1ug of total RNA for each sample by Illumina TruSeq Stranded mRNA Sample Prep Kit (Illumina, Inc., San Diego, CA, USA, #20020595). The first step in the workflow involves purifying the poly‐A containing mRNA molecules using poly‐T‐attached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using SuperScript II reverse transcriptase (Invitrogen, #18064014) and random primers. This is followed by second strand cDNA synthesis using DNA Polymerase I, RNase H and dUTP. These cDNA fragments then go through an end repair process, the addition of a single 'A' base, and then ligation of the adapters. The products are then 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). Indexed libraries were then submitted to an Illumina NovaSeqX (Illumina, Inc., San Diego, CA, USA), and the paired-end (2×100 bp) sequencing was performed by the Macrogen Incorporated.