MGRC Sequencing Sample Submission Guidelines The quality of the data generated by your sequencing project is directly impacted by the quality of the DNA that is used to make the sequencing library. Please follow these sample preparation guidelines to ensure best results. 1.0 DNA Requirements DNA should be extracted using a protocol to preserve the high molecular weight fragments. The best DNA clean-up method is phenol/chloroform extraction, followed by ethanol precipitation with several washes. We recommend carrying out an RNAse-treatment on DNA samples. For each sample, a minimum of 5µg high quality genomic DNA is required. Genomic DNA should be dissolved in TE buﬀer at a minimum concentration of 500ng/µl. It is recommended that you determine the DNA concentration using a ﬂuorometric method such as the Qubit. Determining concentration by using spectrophotometric methods may deliver imprecise nucleic acid concentration. The DNA should have an OD 260/280 ratio of approximately 1.8 and a 260/230 ratio >_ 2. The DNA should be double stranded, not degraded and contain no particular matter. 1.1 DNA for Exome Sequencing If you have requested exome sequencing, the minimum DNA required for exome capture is 10µg of genomic DNA. 1.2 DNA Requirement for Mate-Pair Library If you have requested a mate-pair library, the minimum requirement is 20µg of genomic DNA. 2.0 RNA Requirements Please send us 10µg of high quality total RNA. We recommend the mirVana extraction kit from Ambion, using the protocol for total RNA extraction. Alternatively, for mRNA sequencing, you can send us 200ng of mRNA at a minimum concentration of 2ng/µl. We recommend that a DNase step is carried out during the RNA isolation, and that the RNA is re-suspended in nuclease-free water at a concentration of 500ng/µl or above. The quality of RNA should be assessed using a spectrophotometer, and the 260/280 ratio should be >_ 1.9. If you have an Agilent Bioanalyser please check the total RNA integrity and ensure that the RNA has a RIN (RNA Integrity Number) value greater than 8. Alternatively, a formaldehyde 1% agarose gel can be run and the integrity of RNA judged upon staining with ethidium bromide. High quality RNA shows a 28S rRNA band at 4.5 kb that should be twice the intensity of the 18S rRNA band at 1.9 kb. The RNA will appear as a smear from 0.5-12 kb. 2.1 Ultra-Low Input RNA If you have only limited amounts of RNA sample, we can make mRNA libraries from as little as 100pg RNA. However, the more RNA you can provide, the better the results will be. Please contact us to discuss this option before sending your sample. www.mgrc.com.my 3.0 Shipping of Nucleic Acid A valid study quotation must be signed and received by Malaysian Genomics Resource Centre Berhad prior to any samples being sent. Please email us a copy of the sample submission form and despatch details when you send your samples and be sure to include a printed copy of the sample submission form with the samples. We recommend shipping nucleic acid resuspended in TE buffer, on dry ice. Please ask the courier for the expected length of shipping, and include enough dry ice (2-3 kg per day). As an alternative the nucleic acid can be ethanol-precipitated using the following protocol, and shipped at room temperature. Ethanol Precipitation of DNA Materials required: • 3M sodium acetate, pH 5.2 • DNA • 100% (v/v) ethanol • 70% (v/v) ethanol Procedure: 1. Measure the volume of the DNA sample. 2. If the DNA is in TE buffer, adjust the salt concentration by adding 1/10 volume of 3M sodium acetate, pH 5.2 (final concentration of 0.3M). If the DNA is in a salt-containing solution, adjust the salt concentration to give a final concentration of 0.3M. Mix well but do not vortex. 3. Add 2.5 volumes of cold 100% ethanol. Mix well, do not vortex. 4. Place at -20°C for at least 20 minutes. 5. Spin in a microcentrifuge at maximum speed for 10-15 minutes. 6. Remove supernatant leaving DNA pellet in tube. 7. Add 1ml 70% ethanol, mix, but do not vortex. Spin in a microcentrifuge at maximum speed for 10-15 minutes. 8. Remove the supernatant, leaving the DNA pellet in tube. 9. Add 1ml of 100% ethanol and ship at room temperature. 4.0 Shipping Address Please send your samples to: Sequencing Services, Malaysian Genomics Resource Centre Berhad, 29-10 Level 10 Signature Office, Bandar Mid-Valley, 59200 Kuala Lumpur, Malaysia. www.mgrc.com.my