ctrl 3
Quality of the RNA samples was determined using the Quant-iT™ RiboGreen® RNA Assay Kit (Invitrogen) and Quant-iT™ PicoGreen® dsDNA Assay Kit (Invitrogen) on a Bioanalyzer RNA 6000 Nano Chip (Agilent). Next-generation sequencing library preparation was performed by following TruSeq RNA Sample Preparation v2 protocol (Illumina) with modifications: The mRNA purification step was omitted and instead, 200 ng of total RNA was directly added to the elute-fragment-prime step. The PCR amplification step, which selectively enriches for library fragments that have adapters ligated on both ends, was performed according to the manufacturer’s recommendation but the number of amplification cycles was reduced to 12. Each library was uniquely tagged with one of Illumina’s TruSeq LT RNA barcodes to allow library pooling for sequencing. Library quantitation was performed using Invitrogen’s Picogreen assay and the average library size was determined by running the libraries on a Bioanalyzer DNA 7500 chip (Agilent). Library concentration was normalized to 4 nM and the concentration was validated by qPCR on a ViiA-7 real-time thermocycler (Applied Biosystems), using KAPA Library Quantification Kit Illumina® platforms.  Libraries were then pooled at equal volumes and sequenced in one lane of an Illumina HiSeq2500 rapid run at a final concentration of 11 pM and a read-length of 101 bp paired-end.