Sample Prep

General information for researchers submitting DNA/RNA samples or sequencing libraries to the UCLA Neuroscience Genomic Core

Sept 2014 Version 15

Samples should be plated in PCR style plates or PCR strip tubes. Samples should be arranged by column with no skipped wells (or skipped columns). Please see project specific requirements regarding volume, concentration and, in some cases, plate types below.

Please note that samples submitted to the UNGC may be exhausted during processing. We do not recommend submitting your entire stock of any sample. Any leftover samples will be discarded without additional notification unless you make specific arrangements for their return. A Fedex account number is required to ship any leftover samples.

You must submit a service request and have received a confirmation email before shipping or submitting any samples to the UNGC

The UNGC is a not for profit core facility and our rates are set to cover our operating costs only. All care is taken to ensure that your reactions are completed successfully, however, repeats of failed reactions are not built into the cost of your project. Please budget accordingly.

By utilizing the services of the UNGC you are required to acknowledge the contribution of the Semel Institute UCLA Neurosciences Genomics Core in any publications that result.

Reagents for custom genotyping projects can take up to 6 weeks to arrive. Please plan accordingly.

Genomic DNA for array based Genotyping or Methylation assays should be prepared according to the following guidelines:

20 ul @ 100 ng/ul (50 ng/ul minimum)

Liquid DNA samples should be normalized in 1 X TE to a standard concentration between 50 and 100 ng per ul. Samples with concentrations below 50 ng per ul will not be accepted for genotyping and will be returned to you to be concentrated or re-aliquoted. A minimum volume of 20 ul is required. Please arrange samples by column and avoid skipping wells unless you’re leaving empty wells for control DNA.

We require that DNA samples be sent in 96 well format PCR type plates of these specific types:

96 well skirted v-bottom microplates Cat# MSP-9601 or HSP-9601

from Biorad. Deep well plates (AbGene 0.8 ml MIDI plates (AB-0859) are also acceptable but a minimum of 25 ul is required for this plate type.

If shipping via FedEx or other courier service, please ship samples frozen on dry ice and use strip caps or heat seals to seal the plates. Please download our customs declaration template if shipping from outside of the United States.

If contemplating using Whole Genome Amplification products as samples, please inquire prior to initiating your project.

Total RNA for Array Based Gene Expression analysis should be prepared according to the following guidelines:

20 ul @ 50 ng/ul (20 ng/ul minimum) for Ambion amps

20 ul @ 500 pg/ul (100 pg/ul minimum) for Nugen amps

RIN > 7.0

Input RNA quality is the most significant factor in overall data quality. Therefore it is highly recommended that all total RNA samples be free of all contaminants (Trizol etc) and be assessed for quality using an Agilent Bioanalyzer (or similar). Total RNA submitted for array based gene expression should have a RIN of 7 or higher. We require a minimum volume of 20 ul at a mimimum concentration of 20 ng perl ul. Larger volumes or higher concentrations are helpful. We will confirm the concentration of all samples using a ribogreen assay. Gene expression samples should be in microplates or in multi-channel pipette accessible strip tubes. All plates and or strips must be clearly labeled with your name or other identifying information. Samples should be arranged by column and you should avoid skipping wells or columns.

Low mass (500 pg to 50 ng total mass) samples requiring Nugen amplification should be at least 100 pg per ul, 20 ul total volume. Please note that we are not able to effectively QC extremely low mass samples. Low mass samples will be used as is.

Validated DNA libraries for Sequencing on HiSeq 2500 sequencers should be prepared according to the following guidelines:

50 ul @ 10nM (2nM minimum)

Libraries should be validated with an Agilent Bioanalyzer 2100, 2200 TapeStation or similar. Quantification should be done with PicoGreen or similar fluorescent assay. NanoDrop is not recommended for library quantification. Required volume and concentration: 50 ul minimum at 10nM in Qiagen EB buffer. Libraries should be submitted “ready to run” with all pooling, removal of adapter dimmer and any size selection steps completed beforehand.

Please note that neither long term storage nor analysis of sequencing data is provided by the UNGC. Sequencing runs require either 2 or 8 lanes all of the same run type. Unless submitting enough libraries to fill a flowcell, the timing of your run will be determined by the availability of other libraries to fill existing lanes. You may be required to run a control lane and/or spike in up to 25% phiX control library.

Total RNA or genomic DNA for generating sequencing libraries using TruSeq, TruSeq with RiboZero ribosomal reduction or Nextera chemistry should be prepared according to the following guidelines:

20 ul @ 50 ng/ul (20 ng/ul minimum)

RIN > 8.0 ( for RNA)

Samples should be normalized in nuclease free water and delivered in aliquots of 20 ul minimum volume and 50 ng per ul minimum concentration. Quantification should be done with PicoGreen or similar fluorescent assay. NanoDrop is not recommended for sample quantification. Input RNA quality is the most significant factor in overall data quality. Therefore it is highly recommended that all total RNA samples be free of all contaminants (Trizol etc) and be assessed for quality using an Agilent Bioanalyzer (or similar). Total RNA submitted for RNASeq gene expression should have a RIN of 8 or higher. Samples with RIN values lower than 8.0 are still suitable for processing with RiboZero ribosomal RNA reduction.

We require that samples be sent in clearly labeled PCR strip tubes or in 96 well format skirted PCR type plates. The plates listed below are preferred.

96 well skirted v-bottom microplates Cat# MSP-9601 or HSP-9601

from Biorad. Individual tubes are not accepted for library prep samples

Total RNA for generating sequencing libraries using Nugen Ovation Ultra low mass library prep chemistry should be prepared according to the following guidelines

20 ul @ 500 pg/ul (100 pg/ul minimum)

Samples should be normalized in nuclease free water to between 100pg and 20ng per ul and 20ul total volume. Please note that we are not able to effectively QC extremely low mass samples. Samples will be used as is.

We require that samples be sent in clearly labeled PCR strip tubes or in 96 well format skirted PCR type plates. The plates listed below are preferred.

96 well skirted v-bottom microplates Cat# MSP-9601 or HSP-9601

from Biorad. Individual tubes are not accepted for library prep samples

Please note that we are not able to effectively QC extremely low mass samples. Low mass samples will be used as is.

Total RNA for generating small (miRNA) sequencing libraries using TruSeq Small RNA library prep chemistry should be prepared according to the following guidelines:

20 ul @ 200 ng per ul

Samples must be normalized in nuclease free water to 1.1 ug total mass at 200 ng per ul. Quantification should be done with PicoGreen or similar fluorescent assay. NanoDrop is not recommended for sample quantification.

We require that samples be sent in clearly labeled PCR strip tubes or in 96 well format skirted PCR type plates. The plates listed below are preferred.

96 well skirted v-bottom microplates Cat# MSP-9601 or HSP-9601

from Biorad. Individual tubes are not accepted for library prep samples

Total RNA or gDNA for QC ONLY (Agilent Tapestation Bioanalyzer and/or Pico/RiboGreen quantitation)

6 ul @ 100pg/ul to 500ng/ul (RNA)

6 ul @ 100pg/ul to 50ng/ul (DNA)

Shipping notes:

Samples shipped domestically should be shipped frozen with a sufficient amount of dry ice to last at least three days. Please reference the project ID in the shipping documentation. Please provide a tracking ID for your shipment so that we can monitor its progress. Samples being shipped from overseas: We strongly recommend that you ship your samples with Fedex. Fedex has UCLA power of attorney for customs purposes and this will greatly minimize the chances of your samples being significantly delayed at the US port of entry. Please include several copies of the customs declaration template along with your shipping documentation and inside the container. Please note that a proforma invoice is generally required and must include monetary values for the shipment as line items. If you do experience a customs delay you can contact:

Ellen Laramie
AMERICAN CARGOSERVICE INC.
7880 Convoy Court, San Diego, CA 92111, USA
TEL: +1 (858) 565-4125 FAX: +1 (858) 565-7623
www.acssan.com
Skype: acs.ellen

UNGC shipping info:

Email: jdeyoung@mednet.ucla.edu
Phone: 1 310 825-2390 (no voicemail)

Mailing address:

UCLA Gonda building
695 Charles Young Dr. South
Los Angeles, California
90095