Illumina HiSeq 2000 paired end sequencing
Total RNA from Anas platyrhynchos domestica embryo fibroblasts grown in tissue culture was provided to Source Bioscience (Nottingham, UK) who carried out the library preparation and sequencing. The libraries were prepared using the Illumina TruSeq RNA Sample Preparation kit. The mRNA in the total RNA was purified using poly-T oligo-attached magnetic beads to pull down the poly-A mRNA. After purification, the mRNA was fragmented and copied into first strand cDNA using reverse transcriptase and random primers. This was followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. The newly formed cDNA goes through a process of end repair, the addition of a single 'A' base and the ligation of the adapters. The samples that contain the adapters are selectively enriched for using PCR to create the final library. The libraries were validated using the Agilent BioAnalyser 2100. The libraries were clustered on to a HiSeq v3 flow cell using the Illumina cBot and sequenced on the Illumina HiSeq 2000 using a 100 base pair (bp) sequencing run generating 412 million paired-end reads.
Total RNA from Anas platyrhynchos domestica embryo fibroblasts grown in tissue culture was provided to Source Bioscience (Nottingham, UK) who carried out the library preparation and sequencing. The libraries were prepared using the Illumina TruSeq RNA Sample Preparation kit. The mRNA in the total RNA was purified using poly-T oligo-attached magnetic beads to pull down the poly-A mRNA. After purification, the mRNA was fragmented and copied into first strand cDNA using reverse transcriptase and random primers. This was followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. The newly formed cDNA goes through a process of end repair, the addition of a single 'A' base and the ligation of the adapters. The samples that contain the adapters are selectively enriched for using PCR to create the final library. The libraries were validated using the Agilent BioAnalyser 2100. The libraries were clustered on to a HiSeq v3 flow cell using the Illumina cBot and sequenced on the Illumina HiSeq 2000 using a 100 base pair (bp) sequencing run generating 412 million paired-end reads.