mRNA Sequencing Sample Preparation 1. Quality check of total RNA Customers should carry out a quality check of their total RNA by running it out on a 1% agarose gel, and the integrity of RNA judged upon staining with ethidium bromide. High quality, intact RNA will show a 28S rRNA band at 4.5kb, that should be about twice the intensity of the 18S rRNA band at 1.9kb. Both kb determinations are relative to a 1kb ladder. The mRNA will appear as A smear from 0.5-6kb. Completely degraded RNA will appear as a very low molecular weight smear. Customers are to supply 10ug of purified total RNA

mRNA Sequencing Sample Preparation 2. mRNA Purification from Total RNA mRNA is isolated from total RNA by binding the mRNA to a magnetic oligo(dT) bead. mRNA has a polyA tail and will bind to the oligo(dT) bead. mRNA 5’-UCGGAAGCUGAAGUGAUCAGGGUUCAAUAAAAAAAAAAAAA-3’ Magnetic Oligo (dT) bead Total RNA containing mRNA is added to magnetic oligo (dT) beads PolyA Tail 3’-TTTTTTTTTTTTT 5’-UCGGAAGCUGAAGUGAUCAGGGUUCAAUAAAAAAAAAAAAA-3’ 3’-TTTTTTTTTTTTT mRNA is then eluted from the magnetic oligo (dT) beads by heating to 800C for 2 minutes, & transferred in the supernatant to another tube mRNA in supernatant 5’-UCGGAAGCUGAAGUGAUCAGGGUUCAAUAAAAAAAAAAAAA-3’

mRNA Sequencing Sample Preparation 3. Fragmentation of mRNA The mRNA is fragmented into small pieces using divalent cations under elevated temperature. 5’-UCGGAAGCUGAAGUGAUCAGGGUUCAAUAAAAAAAAAAAAA-3’ mRNA Add fragmentation buffer and PCR thermocycle At 700C for 5 minutes Random fragmentation 5’ 3’ 5’ 3’ 5’ 3’ Generates fragments ranging in size from 100 bases to 5000 bases. 5’ 3’ 5’ 3’ 5’ 3’ 5’ 5’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 4. First Strand cDNA Synthesis Random Hexamer Primers The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and a high concentration of random hexamer primers mRNA 5’ 3’ 3’ 5’ cDNA strand

mRNA Sequencing Sample Preparation 5. Second Strand cDNA synthesis Remove the strand of mRNA and synthesize a replacement strand generating double-stranded cDNA. mRNA RNase H 5’-UCGGAAGCUGAAGUGAUCAGGGUUCAAUAAAAAAAAAAAAA-3’ First Strand cDNA 3’-AGCCTTCGACTTCACTAGTCCCAAGTTATTTTTTTTTTTTT-5’ RNase H activity enzymatically degrades the mRNA strand. 3’-AGCCTTCGACTTCACTAGTCCCAAGTTATTTTTTTTTTTTT-5’ DNA Polymerase I activity generates second strand cDNA to form double-stranded cDNA. DNA Polymerase I Second Strand cDNA 5’-TCGGAAGCTGAAGTGATCAGGGTTCAATAAAAAAAAAAAAA-3’ 3’-AGCCTTCGACTTCACTAGTCCCAAGTTATTTTTTTTTTTTT-5’ First Strand cDNA

mRNA Sequencing Sample Preparation 6. Add ‘A’ Bases to the 3’ End of the DNA Fragments P5’-AGTCTTGGATCGAC-3’ 3’-TCAGAACCTAGCTG-5’P An ‘A’ base is added to the 3’ end of the blunt phosphorylated DNA fragments, using the polymerase activity of Klenow fragment (3’ to 5’ exo minus). This prepares the DNA fragments for ligation to the adapters, which have a single ‘T’ base overhang at their 3’ end P5’-AGTCTTGGATCGACA-3’ 3’-ATCAGAACCTAGCTG-5’P 7. Ligate Adapters to the DNA Fragments P5’-AGTCTTGGATCGACA-3’ 3’-ATCAGAACCTAGCTG-5’P Adapters are ligated to the ends of the DNA fragments, preparing them to be Hybridized to the flow cell P5’-AGTCTTGGATCGACA-3’ 3’-ATCAGAACCTAGCTG-5’P

mRNA Sequencing Sample Preparation 8.Purify Ligation Products The products from the ligation are purified on a 2% agarose gel, to remove all unligated adapters, remove any adapters that may have ligated to one another, & select a size range of templates to go on The cluster generation platform Excise a gel slice in the 200bp (+/- 25bp) and purify

mRNA Sequencing Sample Preparation 9. Enrich the Adapter-Modified cDNA Fragments by PCR PCR is used to selectively enrich those cDNA fragments that have adapter molecules on both ends, & to amplify the amount of cDNA in the library. The PCR is performed with two primers that anneal to the ends of the adapters P5’-AGTCTTGGATCGACA-3’ 3’-ATCAGAACCTAGCTG-5’P Denaturation P5’-AGTCTTGGATCGACA-3’ Primer Annealing Amplify using the following PCR protocol: *30 seconds at 980C *15 cycles of: 10 seconds at 980C 30 seconds at 650C 30 seconds at 720C *5 minutes at 720C *Hold at 40C 3’-ATCAGAACCTAGCTG-5’P Extension P5’-AGTCTTGGATCGACA-3’ 3’-NNNNNTCAGAACCTAGCTGTNN-5’ 5’-NNTAGTCTTGGATCGACNNNNN-3’ 3’-ATCAGAACCTAGCTG-5’P

mRNA Sequencing Sample Preparation 10. Validate the Library Perform the following quality control steps on the DNA Library: Determine the library concentration by measuring its absorbence at 260nm. The yield from the protocol should be between 500-1000ng of DNA. Measure the 260/280 ratio. It should be ~1.8-2.0. Either load 10% of the volume of the library on a gel and check that the size range is as expected, or run the DNA library on an Agilent 2100 Bioanalyzer. Sample Determine the molar concentration of the library ready for Cluster Generation Data from Agilent 2100 Bioanalyzer