Small RNA 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

Small RNA Sample Preparation 2. RNA Quality Check by Agilent 2100 Bioanalyzer 18S rRNA 28S rRNA Marker The AWCGS runs an isolated RNA quality check by Agilent 2100 Bioanalyzer system. The Bioanalyzer generates an RNA integrity number (RIN). RNA samples with RIN >8 Will be further processed for Solexa expression analysis.

Small RNA Sample Preparation 3. Isolate Small RNA by Denaturing PAGE Gel 100bp 80bp 60bp 40bp 20bp 10ug of total RNA is run on a 15% TBE-urea gel for 1 hour at 200V with a size ladder in 20bp steps Up to 100bp. Cut out a band of gel corresponding to the 18-30 nucleotide bands in the marker lane, and gel extract.

Small RNA Sample Preparation 4. Ligate 5’ Adapter 5’ small RNA adapters A single stranded 5’ small RNA adapter is ligated to the 5’ end of each small RNA fragment. The 5’ small RNA adapter is necessary for reverse transcription and amplification of the small RNA fragment. This adapter also contains the DNA sequencing primer binding site. Gel purify the adapter modified small RNA Products on a 15% TBE-urea gel, and excise And purify the products in the 40-60nt size range. Small RNA fragments 18-30nt in length 3’ small RNA adapters 5. Ligate 3’ Adapter A single stranded 3’ small RNA adapter is ligated to the 3’ end of each small RNA fragment. The 3’ Small RNA adapter corresponds to the surface bound amplification primer on the flow cell used for Cluster generation. Gel purify the adapter modified small RNA Products on a 10% TBE-urea gel, and excise And purify the products in the 70-90nt size range. 5’ adapter ligated small RNA fragments 5’ & 3’ adapter ligated small RNA fragments

6. Reverse Transcribe and Amplify the Small RNA Ligated with Adapters Small RNA Sample Preparation 6. Reverse Transcribe and Amplify the Small RNA Ligated with Adapters Reverse transcription followed by PCR is used to create cDNA constructs based on the small RNA ligated with 5’ and 3’ adapters. This protocol selectively enriches those RNA fragments that have adapter molecules on both ends. The PCR is performed with two primers that anneal the ends of the adapters. 3’ 5’ 5’ 3’ Purified 5’ and 3’ ligated RNA Reverse Transcription with SuperScript III Reverse Transcriptase – First strand synthesis generating single Strand cDNA 3’ 5’ cDNA strand 5’ 3’ RNA strand RNase OUT denatures RNA strand 3’ 5’ PCR amplification generates second cDNA strand and enriches adapter ligated products 3’ 5’ 5’ 3’

Small RNA Sample Preparation 7. Purify the Amplified cDNA Construct Gel purify the adapter modified cDNA construct on a 6% TBE PAGE gel, and excise and purify the products in the 92bp size range. 25bp ladder is in 25bp Steps up to 300bp 100bp 8. Validate the Library 92bp 75bp Determine the molar concentration of the library ready for Cluster Generation 50bp 25bp Run the products on an Agilent 2100 Bioanalyzer, to check The size, purity, and concentration of the sample. The yield should be 500-100ng of DNA. Measure the OD 260/280 ratio, which should be ~1.8. Run the products on an Agilent 2100 Bioanalyzer, to check The size, purity, and concentration of the sample. The yield should be 500-100ng of DNA. Measure the OD 260/280 ratio, which should be ~1.8. Sample Ladder Cut from gel and purify