**Required information and guidelines for submission of Total RNA and
double-stranded cDNA to the DOE Joint Genome Institute**

The success of an EST project is primarily dependent on the quality of
the total RNA (or in some cases ds-cDNA) received from the collaborator.
Generally we ask for 1-2 RNA samples for smaller genomes (\<50Mb) and
3-4 for larger genomes. For EST-only projects, the number of samples
will vary. The samples should be chosen to represent a wide range of
transcripts. If more RNA samples are desired, consider pooling them.
Please send only one tube per sample. JGI will select the most effective
sequencing technology to process the samples and sequence the ESTs;
generally this will be the 454 Titanium platform. Please contact your
project manager or JGI Project Management
(<jgi_project_management@lanl.gov>) or Erika Lindquist
(<ealindquist@lbl.gov>) with any questions or concerns.

**Information required for submission*:***

1\. Prior to shipment of sample, fill in Collaborator Sample Information
form at https://my.jgi.doe.gov/csi/. You will need to request a myJGI
login to access this form. A *gel image or Bioanalyzer result is
required for RNA/cDNA submissions.*

2\. Wait for approval to ship and then include a printed copy with the
sample shipment.

3\. Complete one form for each sample submitted.

**RNA Sample(s) for Sanger, 454 and Illumina sequencing:**

*Most projects begin with total RNA. The JGI requires 500ug of
nondegraded, noncontaminated, high quality total RNA for each Sanger and
454 library, and 10ug for each Illumina (mRNA-Seq) library.* If you do
not have enough material, JGI may be able to amplify the sample using
one of several methods depending on the goals of your project and the
amount of sample available. Please discuss this with your project
manager.

Different types of samples may require specific RNA isolation
techniques. JGI has successfully used standard methods involving Trizol,
Rneasy and for many samples further purified using LiCl. Contaminants in
the preparation will often cause an overestimated measure of RNA
quantity when using OD measurements. The JGI will QC the samples prior
to library construction and will contact you with any issues.

1\. Total RNA must be submitted in a labeled microfuge tube (1.5-2mL) in
nuclease free water at a concentration of 1ug/uL. **Please ship one tube
per RNA sample.**

2\. RNA must be free of DNA contamination (based on gel or Bioanalyzer
results). Samples should be treated with a standard DNase treatment
prior to submission.

3\. Total RNA must have an OD260/280 ratio of between 1.7-2.0. A lower
ratio is often indicative of protein contamination, a higher ratio may
indicate residual guanidine thiocyanate or beta-mercaptoethanol. Protein
contaminants should be re-extracted using phenol:chloroform:isoamyl
alcohol; other contaminants by EtOH ppt. In general, JGI recommends LiCl
extraction for cleaning up RNA.

4\. RNA must not show signs of degradation as measured by strength of
ribosomal peaks (gel electrophoresis or Bioanalyzer results). Please see
example at the end of the document.

5\. If pooled RNA samples are to be submitted, pool them prior to
shipment. It is recommended that each sample be checked for quality
prior to including it in the pool. Gel photo(s) submitted to JGI should
be for the total pooled sample.

6\. Gels should be run with a standard MW marker to assess the size of
the ribosomal bands. State the sizes of the markers as well as the
estimated size of the Total RNA bands when gel photo is submitted.

7\. Tube label should include Genus, species and sample/tissue name,
plus the numeric sample ID included in the shipping approval email. This
ID will ensure proper tracking of the sample once it arrives.

**ds-cDNA sample(s) for Sanger, 454 and Illumina sequencing:**

For some projects it is necessary for the collaborator to ship double
stranded cDNA (dscDNA) rather than total RNA to the JGI. *Quantity
required is 3 ug minimum for Sanger and Illumina, 5 ug for 454.*

Format for shipping:

a.  Resuspended in TE (10 mM Tris, ph 7.5 -- 8, EDTA 0.1 mM at \>= 50
    ng/ul shipped on dry ice

b.  *Only one tube per sample*, labeled as described above.

In order to efficiently sequence cDNA using new technologies, the dscDNA
template fragments should range in size between 300-800 bases for 454
sequencing and shorter for other short read technologies (contact JGI
Project Management). In order to assure this size range, the standard
JGI 454 protocol for cDNA shotgun sequencing involves generation of
first strand cDNA, nick translation to generate the second strand,
fragmentation of the dscDNA, size selection, end repair, ligation of
short read technology adaptors, quantitation and finally sequencing. JGI
will consider sequencing collaborator provided dscDNA samples. In
general, dscDNA samples may be submitted in 3 forms; fragmented with or
without adaptors ligated (454 and short read technologies) or
unfragmented (454 and short read technologies). *Please contact JGI
Project Management to discuss proposed strategy and protocols.*

Fragmented dscDNA:

Fragmented dscDNA lengths for 454 and short read technology sequencing
must be in appropriate ranges (see above) and can be achieved by methods
not limited to but including sonication, nebulization and random primed
cDNA generation. Fragmented dscDNA should be end-repaired and gel
purified before shipping. Please contact JGI Project Management for
details regarding adaptor ligation. Refer to the quantity and quality
measurements described above. Please indicate as much information as
possible describing methods used for sample preparation, fragment size
range, etc in the CSI form.

Unfragmented dscDNA:

High quality dscDNA generated should yield a broad range of transcript
lengths. JGI will process the sample according to the standard protocol.
Refer to the quantity and quality measurements below. Please indicate
methods used for preparation of the sample in the appropriate fields in
the RI.

**Sample gel images and Bioanalyzer results**

1\. Gel reference from the Ambion website
(<http://www.ambion.com/techlib/tn/83/8313.html>)

![](media/image1.png){width="2.10625in" height="3.029166666666667in"}

2\. Bioanalyzer results of RNA and dscDNA:

Total RNA:

> \- clear 18s and 28s rRNA peaks, no degradation.

![](media/image2.pct){width="3.1256944444444446in"
height="1.3743055555555554in"}

PolyA RNA:

\- limited residual rRNA, broad distribution of transcripts

![](media/image3.pct){width="3.0in" height="1.4201388888888888in"}

> Unfragmented dscDNA:
>
> \- broad distribution of transcripts
>
> ![](media/image4.pct){width="2.875in" height="1.2743055555555556in"}