GSM9313009: VCaP_100nM_ARi_p300i_12hr_Rep1; Homo sapiens; RNA-Seq
Total RNA was extracted from cells using QIAzol Lysis Reagent (QIAGEN). Initially, 1x10^6 cells were lysed with 700 µL of QIAzol. The lysates were incubated at room temperature for 5 minutes to dissociate nucleoproteins, then mixed with 140 µL chloroform and centrifuged at 12,000 g for 15 minutes at 4°C to achieve phase separation. The aqueous phase was then mixed with 1.5x volume of ethanol and applied to a RNeasy Mini spin column (QIAGEN) for RNA purification, following the miRNeasy Mini Kit protocol. The RNA was then eluted in RNase-free water, and its concentration and purity assessed using a NanoDrop spectrophotometer. RNA-seq libraries were prepared using 200–1,000 ng of total RNA. PolyA+ RNA isolation, cDNA synthesis, end-repair, A-base addition, and ligation of the Illumina indexed adapters were performed according to the TruSeq RNA protocol (Illumina). Libraries were size selected for 250–300 bp cDNA fragments on a 3% Nusieve 3:1 (Lonza) gel, recovered using QIAEX II reagents (QIAGEN), and PCR amplified using Phusion DNA polymerase (New England Biolabs). Library quality was measured on an Agilent 2100 Bioanalyzer for product size and concentration. Paired-end libraries were sequenced with the Illumina HiSeq 2500, (2 × 100 nucleotide read length) with sequence coverage to 15–20M paired reads.