Agilent bioanalyzer rna nano kit

Quality and quantity of the 60 nucleic acid samples were established using the Agilent Bioanalyzer RNA Nano kit and the Nanodrop 1000, respectively. GeneChip 3′ IVT PLUS Reagent Kit (Thermo Scientific) provided sufficient amplified double-stranded cDNA for each holoclone, meroclone, or paraclone total RNA sample, letting to in vitro transcribe it to labeled cRNA, that could be fragmented and therefore hybridized onto single GeneChip™ Human Genome U133 Plus 2.0 Array (ThermoScientific), following the manufacturer’s indications. To avoid batch effect among samples, i.e. no technical sources of variation added to the samples during handling, samples were randomized during RNA isolation, sample preparation, and hybridization/scanning working sessions. All samples were processed with the same reagents lot number, when available. Sample and hybridization quality controls were carried out with Transcriptome Analysis Console (TAC, ThermoScientific) to verify complete and unbiased coverage of the transcriptome.

From each dissection of E13.5 cortex or basal ganglia, RNA was isolated using QIAGEN RNeasy mini columns. RNA was treated with Turbo DNase and inactivated. RNA quality was assessed using Agilent Bioanalyzer RNA Nano kit, and samples with RNA Integrity Number values greater than 9.0 were used for subsequent profiling. RNA-seq libraries were generated using Nugen’s Ovation Mouse RNA-seq kit and amplified for 15 cycles. Libraries were quantified using Agilent Bioanalyzer High Sensitivity DNA kit, and sequenced on Hiseq 2500 using paired end sequencing.