GSM4845017: A/64PTX-30d - rep 1; Homo sapiens; RNA-Seq
Total RNA was isolated from 5x106 cells using RNeasy Mini Kit (Qiagen) according to manufacturer instructions. Method is based on lysing cells in RLT buffer containing guanidine isothiocyanate, purification the product on spin column with silica-gel membrane and treatment by DNaseI to remove genomic DNA contamination. Isolated RNA was stored in -80ºC. RNA integrity number (RIN) was analyzed using Bioanalyzer 2100 and RNA 6000 Nano Kit (Agilent Technologies), according to manufacturer instructions. RNA concentration was measured using fluorometer Qubit and Qubit RNA BR Assay Kit (Life Technologies), according to manufacturer instructions. Fourteen NGS libraries were prepared following the protocol  of the TrueSeq RNA Sample Preparation Kit v2 (Illumina). From 4 μg of total RNA input of each sample the poly-A containing mRNA molecules were purified using oligo-dT attached magnetic beads. Then the mRNA was fragmented into about 150 nucleotide (nt) pieces using divalent cations under 65ºC. The cleaved mRNA fragments were primed with random hexamers and reverse transcribed by SuperScript III reverse transcriptase into first strand cDNA. Subsequent stage involved second strand cDNA synthesis using DNA Polymerase I and removing the RNA template using RNaseH. Double stranded cDNA (ds cDNA) was purified using AMPure XP beads. Then the overhangs resulting from fragmentation were converted into blunt ends, phosphorylated on 5' ends and a single A nucleotide was added to 3' ends. Multiple indexing adapters with single T nucleotide 3' ends were ligated to the ends of the ds cDNA. Finally, the 12 cycle PCR was performed to selectively enrich those DNA fragments that had adapter molecules on both ends and to amplify the amount of DNA to sequence. Libraries were qualified using Bioanalyzer 2100 and DNA 1000 Kit (Agilent),  according to manufacturer instructions. In all libraries the final product was an anticipated band at approximately 270 bp. The total concentration of the libraries was measured using fluorometer Qubit and Qubit dsDNA BR Assay Kit (Life Technologies), according to manufacturer instructions. Libraries were additionally quantified with qPCR according to Illumina recommended protocol, using Rotor-Gene Q (Qiagen) and KAPA SYBR FAST qPCR Master Mix Universal (Kapa Biosystems). Validated libraries were diluted to 10 nM concentration, then pooled in batches of two differentially indexed samples in equal amounts, denatured and diluted to 10 pM concentration. Cluster generation was performed in cBot (Illumina), where the libraries were bound with a flow cell (two libraries per lane). Sequencing by synthesis (SBS) was performed with Genome Analyzer IIx (Illumina). Technical replicates of the libraries were clustered and sequenced with the use of different flow cell, but with the same 75-cycle multiplexed single-read sequencing recipe. Finally, we obtained approximately 25 million 75 nt-long reads per library.