Illumina HiSeq 2000 paired end sequencing; RNAseq of wild-type and giantin knockout retinal pigment epithelial cell lines
RNAseq of wild-type and giantin knockout retinal pigment epithelial cell lines
RNA from cultured cells Nucleospin RNA kit from Machery-Nagel The libraries were constructed by The Earlham Institute on a PerkinElmer Sciclone using the TruSeq RNA protocol v2 (Illumina 15026495 Rev.F). The library preparation involved the initial QC of the RNA using a Tecan plate reader with the Quant-iT™ RNA Assay Kit (Life technologies/Invitrogen Q-33140) and the Quant-iT™ DNA Assay Kit, high sensitivity (Life technologies/Invitrogen Q-33120). Finally, the quality of the RNA was established using the PerkinElmer GX with a high sensitivity chip and High Sensitivity DNA reagents (PerkinElmer 5067-4626). RNA quality scores were 8.7 and 9.8 for two of the samples and 10.0 (for the remaining 4 samples). 1 ug of RNA was purified to extract mRNA with a poly- A pull down using biotin beads, fragmented and first strand cDNA was synthesised. This process reverse transcribes the cleaved RNA fragments primed with random hexamers into first strand cDNA using reverse transcriptase and random primers. The ends of the samples were repaired using the 3' to 5' exonuclease activity to remove the 3' overhangs and the polymerase activity to fill in the 5' overhangs creating blunt ends. A single ‘A’ nucleotide was added to the 3’ ends of the blunt fragments to prevent them from ligating to one another during the adapter ligation reaction. A corresponding single ‘T’ nucleotide on the 3’ end of the adapter provided a complementary overhang for ligating the adapter to the fragment. This strategy ensured a low rate of chimera formation. The ligation of a number indexing adapters to the ends of the DNA fragments prepared them for hybridisation onto a flow cell. The ligated products were subjected to a bead based size selection using Beckman Coulter XP beads (Beckman Coulter A63880) to remove un-ligated adapters, as well as any adapters that may have ligated to one another. Prior to hybridisation to the flow cell the samples were amplified by PCR to selectively enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. The PCR was performed with a PCR primer cocktail that annealed to the ends of the adapter. The insert size of the libraries was verified by running an aliquot of the DNA library on a PerkinElmer GX using the High Sensitivity DNA chip and reagents (PerkinElmer CLS760672) and the concentration was determined by using the Tecan plate reader. The resulting libraries were then equimolar pooled and Q-PCR was performed on the pool prior to clustering.