GSM6407691: PC3_2; Homo sapiens; OTHER
Total RNA extraction for HCT-116, MCF-7, PC3 and TOV-112D cell lines was performed using the RNeasy mini kit (Qiagen) according to the manufacturer's instructions, using on column DNase digestion with RNase-Free DNase Set (Qiagen). To keep smaller RNA molecules, 1.5 volumes Ethanol 100% were used instead of the 1 volume of ethanol 70% recommended. Agilent 2100 Bioanalyzer was used to assess the quality of the RNA of each sample. 2 ug of DNAse treated RNA was ribodepleted using Ribo-Zero Gold (Illumina), following the manufacturer's instructions. The resulting rRNA free RNA was purified with RNA Clean and Concentrator (RCC) kit (Zymo Research) using a slightly modified protocol (400 ul ethanol 100% per 50 ul sample) allowing us to retain RNA ≤ 80 nucleotides, followed by a fragmentation for 2-4 min using NebNext Magnesium RNA Fragmentation Module (New England Biolabs). The sample was purified once again with the RCC kit and was then dephosphorylated using T4 Polynucleotide Kinase (Epicentre) followed by an additional purification with the RCC kit. For cDNAs preparation, we used 1 uM TGIRT-III reverse transcriptase (Ingex, LLC) for 15 minutes at 60 °C, which also permits the binding of an Illumina Read 2 sequencing primer DNA complement to the 5' end of the cDNA. Next, an Illumina Read 1 sequencing adenylated DNA oligonucleotide complement was ligated to the 3' end of the cDNA using Thermostable 5' AppDNA / RNA Ligase (New England Biolabs). The resulting product was amplified using a 12 cycle PCR reaction in order to synthesize the second strand and add sequences for Illumina flowcell capture and indexing. A two-round purification step was performed using Ampure XP beads (Beckman-Coulter). The quality of the product was assessed on a 2100 Bioanalyzer (Agilent). The pooled libraries were sequenced on a NextSeq 500 platform (Illumina) (2 x 75 nt) using a NextSeq500 High Output Kit v2.5 (150 cycles) (Illumina). HCT-116, MCF-7, PC3 and TOV-112D were all sequenced in two replicates together multiplexed in one sequencing run.