Illumina whole genome expression microarray

Amplification of sample RNA for the Illumina array was achieved by using the Illumina® _{TotalPrep RNA Amplification Kit (AMIL1791) as per manufacturer’s}

instructions. The kit generates biotinylated, amplified RNA for hybridization with Illumina arrays. The RNA amplification protocol is based on the method developed in the laboratory of James Eberwine (Van Gelder et al., 1990). The method included reverse transcription with an oligo(dT) primer bearing a T7 promoter using Array Script, a reverse transcriptase (RT) designed to yield higher concentrations of first strand cDNA than wild-type enzymes which resulted in the synthesis of virtually full-length cDNA. The cDNA was then subjected to second strand synthesis and cleanup, where it became a template for in vitro transcription (IVT) with T7 RNA polymerase. IVT along with biotin-UTP was used to generate hundreds to thousands of biotinylated copies of antisense RNA. This labelled antisense RNA (cRNA; aRNA) was then hybridised to Illumina Beadchips.

Starting RNA was subjected to QC checks by NanoDrop to ensure no contaminating proteins, DNA, phenol ethanol and salts remained after miRneasy RNA isolation. The Agilent® _{2100 bioanalyser was employed to ensure RNA}

sample integrity. RNA Integrity Number (RIN) of 8.0+ was deemed acceptable for samples to go forward for microarray analysis. In a non-stick, sterile, RNase-free tube, 350 ng of Total RNA (50-500 ng is recommended) was brought to 11 µL with nuclease-free water. Separately, Reverse Transcription Master Mix was assembled at RT in a nuclease-fee tube. For a single 20 µL reaction, 1 µL of T7 Oligo(dT) Primer, 2 µL of 10X First Strand Buffer, 4 µL of dNTP Mix, 1 µL of RNase Inhibitor and 1 µL of ArrayScript were mixed gently, centrifuged briefly and placed on ice. 9 µL of Reverse Transcription Master Mix was transferred to each RNA sample and the sample mixed by pipetting up and down 2-3 times then

flicking the tube 3-4 times with brief centrifugation to bring the sample to the bottom of the centrifuge tube. The reactions were then placed in a thermal cycler set to 42°C for 2 h, after which the samples were centrifuged and placed on ice. On ice, a Second Strand Master Mix was prepared in a nuclease-free tube comprising, in order, 63 µL nuclease-free water, 10 µL 10X Second Strand Buffer, 4 µL dNTP Mix, 2 µL DNA Polymerase and 1 µL RNase H for a single 100 µL reaction. This was mixed gently and briefly centrifuged. 80 µL of Second Strand Master Mix was added to each sample, the sample mixed by pipetting up and down 3-4 times and centrifuged briefly, put in a thermal cycler set to 16°C for 2 h and placed on ice. 250 µL of cDNA Binding Buffer was added to each sample, mixed by pipetting up and down 2-3 times and flicking the tube 3-4 times with brief centrifugation and the sample loaded onto the cDNA Filter Cartridge. The cDNA cartridge was centrifuged at 10000 x g for 1 minute at RT and the flow- through discarded. 500 µL Wash Buffer was added to the cDNA Filter Cartridge and centrifuged at 10,000 x g for 1 minute with the flow-through discarded and the empty cDNA Filter Cartridge centrifuged for an additional minute to remove all traces of Wash Buffer. The cDNA Filter Cartridge was transferred to a cDNA Elution Tube. 20 µL of pre-heated nuclease-free water at 55°C was added to the centre of the filter in the cDNA Filter Cartridge and left at RT for 2 min. The cDNA Filter Cartridge was centrifuged for 1 minute at 10,000 x g, leaving the double stranded cDNA in the eluate. The cDNA was stored overnight at -20°C at this point, as directed by the protocol. Each cDNA sample was transferred to a 0.5 mL nuclease-free microcentrifuge tube and at RT an IVT Master Mix assembled, comprising 2.5 µL T7 10X Reaction Buffer, 2.5 µL T7 Enzyme Mix and 2.5 µL Biotin-NTP Mix per 25 µL reaction. This was mixed gently, centrifuged briefly and placed on ice. 7.5 µL of IVT Master Mix was added to each cDNA sample, mixed by pipetting up and down 2-3 times and flicking the tube 3-4 times with brief centrifugation, placed in a thermal cycler set to 37°C for 14 h. The reaction was stopped by the addition of 75 µL Nuclease-free water to each cRNA sample, bringing the final volume to 100ul. 350 µL of cRNA Binding Buffer was added to each cRNA sample, 250 µL of ACS reagent grade 100% ethanol added to sample and the mixture pipetted up and down 3 times. The mixture was immediately added to the centre of the filter of a cRNA Filter Cartridge, centrifuged at 10,000 x g for 1 minute and the flow-through discarded. 650 µL Wash Buffer was then added to the cRNA Filter Cartridge and centrifuged at 10,

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000 x g for 1 minute, the flow-through discarded and the cRNA Filter Cartridge centrifuged for an additional minute to remove all trace of Wash Buffer. The cRNA Filter Cartridge was transferred to a fresh cRNA Collection Tube, 200 µL nuclease-free water heated to 55°C added to the centre of the filter in the cRNA Filter Cartridge and placed in a hybridisation oven at 55°C for 10 min. The cRNA Filter Cartridge was centrifuged at 10, 000 x g for 1.5 min, eluting the cRNA. At this stage samples were subjected to Nanodrop analysis to ascertain cRNA concentration was at least 150ng/µL, if not then samples were concentrated by vacuum centrifugation.

The resultant samples were then hybridised to the Illumina® HumanHT-12 v3 Expression BeadChip as per manufacturers protocol (cat number BD-901-1002). Briefly, the cRNA was pre-heated to 65°C for 5 min, vortexed, pulse centrifuged at 250 x g and allowed to cool to RT and 750ng pipetted into a microcentrifuge tube with the appropriate volume of nuclease-free water added to bring the sample to 5 µL. 10 µL of HYB was added to the sample and the Hyb Chamber assembled with 200 µL of HCB in each humidifying buffer reservoir. The sample was pipetted onto the centre of each inlet port on the beadchip, the beadchip placed into the Hyb chamber and the Hyb chamber placed into an illumina Hybridisation over at 58°C for 14 h. The beadchips were removed from the Hyb chambers, submerged in diluted wash E1BC buffer and the cover-seal removed. Following this the beadchips were placed in a slide rack submerged in a glass staining dish containing diluted wash E1BC then transferred to a Hybex Waterbath containing High-Temp Wash buffer at 55°C for 10 min. The slide rack was placed into a staining dish containing 250 mL fresh Wash E1BC buffer and plunged in and out of the solution 5-10 times before being put back into the staining dish and placed on an orbital shaker for 5 min at RT at medium speed. The beadchips were subsequently moved to a new staining dish containing 250 mL fresh 100% Ethanol, the sliderack plunged in and out of the solution 5-10 times and the staining dish placed on an orbital shaker for 10 min at RT. The beadchips were transferred back to the original staining dish containing 250 mL Wash E1BC buffer, plunged in and out of the solution 5-10 times and placed on an orbital shaker for 2 min at RT. The beadchips were subsequently put face up into a beadchip wash tray with 4 mL Block E1 buffer and placed on a rocker mixer at medium speed for 10 min at RT. The beadchips were transferred to

another wash tray containing 2 mL Block E1 buffer and 1:1,000 dilution of Cy3- Streptavidin and placed on a rocker mixer at medium speed for 10 min at RT. Subsequently, the beadchips were transferred to fresh Wash E1BC, plunged in and out of the solution 5 times before being placed submerged in Wash E1BC and placed on an orbital shaker at medium-low for 5 min at RT. The slide rack containing the beadchips was then transferred to a centrifuge and subjected to 1,400 x rpm for 4 min at RT after which they were imaged on an Illumina® BeadArray Reader.

Whole genome gene expression was determined for SA461 hESC differentiating to endothelial lineage as per design of the miRNA microarray, with data quantile normalised and exported from Genome Studio (Illumina, CA, USA) for uploading to Partek (Partek Inc., MO, USA) where the data was subjected to transformation by Principle Components Analysis (PCA). An analysis of variance (ANOVA) model was then created with step-up false discovery rate (FDR), taking into account chip effects and allowing pairwise comparisons. The data were then uploaded to Ingenuity Pathway Analysis (IPA, CA, USA, www.ingenuity.com) and pathways with the most significantly different genes interrogated, as well as dynamic expression of genes expressed at specific timepoints. In addition, a primary bivalency dataset from H9 hESC (Ku et al., 2008) was integrated with the data from the Illumina, allowing a prediction of bivalent status to be made for around ~2,500 genes.