cDNA Library Creation Protocol

Version Number: 1.0

> Production Dates: 03/16/2007
>
> Version Date: 04/02/2007
>
> Author: Zhiying (Jean) Zhao, Dean Ng,
>
> Reviewed/Revised by: Erika Lindquist, [Paul
> Richardson](http://www/employees/directory_hits.php?searchstring=pmrichardson),
> Feng Chen

# Summary

This protocol describes the creation of a cDNA library for 454. mRNA is
reverse transcribed with SuperScriptIII using dT~15~VN~2~ primer. cDNA
is synthesized with *E. col*i DNA Ligase , *E. coli* DNA polymerase I ,
and *E coli* RNaseH. cDNA is then fragmented by sonication. The cDNA is
then ready for 454 sstDNA preparation (start from protocol "GS20 DNA
Library Preparation" step2).

# QC criteria

Starting material

Quantity - amount of polyA RNA isolated from sample in ug.

2-4ug polyA RNA with %rRNA \<20%(typical \<10%).

Ethanol precipitate RNA to concentrate samples to \>200 ng/ul, if
necessary.

Use NanoDrop or Agilent Bioanalyzer to verify RNA concentration.

> ![](media/image1.pct){width="2.30625in" height="1.4013888888888888in"}
>
> Figure: PolyA enriched RNA

Finished product:

Should have \>1ug fragment cDNA, size range from 100-800bp, as
determined by Bioanalyzer.

# Materials and Reagents -- cDNA Synthesis 

+----------------------------+----------------------+-----------------+
| [Materials/Reage           | [Vendor]{.underline} | [Stock          |
| nts/Equipment]{.underline} |                      | Numb            |
|                            |                      | er]{.underline} |
+----------------------------+----------------------+-----------------+
| *                          |                      |                 |
| *[Materials]{.underline}** |                      |                 |
+----------------------------+----------------------+-----------------+
| 0.2 ml PCR Tubes           | PGC                  | 16-7038-30      |
+----------------------------+----------------------+-----------------+
| Pipette Tips, 1000ul, LTS, | Rainin               | RT-L1000F       |
| Filtered                   |                      |                 |
+----------------------------+----------------------+-----------------+
| Pipette Tips, 200ul,       | Rainin               | RT-L200F        |
| Filtered                   |                      |                 |
+----------------------------+----------------------+-----------------+
| Pipette Tips, 20ul, LTS,   | Rainin               | RT-L10F         |
| Filtered                   |                      |                 |
+----------------------------+----------------------+-----------------+
| Phase lock gel **light**   | Eppendorf            | 955154169       |
| (1.5ml tube, 200/pack)     |                      |                 |
+----------------------------+----------------------+-----------------+
| **                         |                      |                 |
| *[Reagents]{.underline}*** |                      |                 |
+----------------------------+----------------------+-----------------+
| Nuclease-free H~2~O        | Ambion               | AM9930          |
| (500ml)                    |                      |                 |
+----------------------------+----------------------+-----------------+
| Bioanalyzer DNA 1000       | Agilent              | 5067-1505       |
| Reagents                   |                      |                 |
+----------------------------+----------------------+-----------------+
| Bioanalyzer DNA 7500       | Agilent              | 5067-1507       |
| Reagents                   |                      |                 |
+----------------------------+----------------------+-----------------+
| 5x first strand buffer     | Invitrogen           | 18080-044       |
|                            |                      |                 |
| 0.1M DTT                   |                      |                 |
|                            |                      |                 |
| SuperscriptIII (200u/ul,   |                      |                 |
| 10,000u)                   |                      |                 |
+----------------------------+----------------------+-----------------+
| 5X second Strand Buffer    | Invitrogen           | 10812-014       |
| (0.5ml)                    |                      |                 |
+----------------------------+----------------------+-----------------+
| 10mM dNTP Mix (100ul, 1ml) | Invitrogen           | 18427-013,      |
|                            |                      | 18427-088       |
+----------------------------+----------------------+-----------------+
| 10mM dNTP Mix (200ul, 1ml) | MBI Fermentas        | R0191, R0192    |
+----------------------------+----------------------+-----------------+
| *E. coli* DNA Ligase (10   | Invitrogen           | 18052-019       |
| U/ul, 100U)                |                      |                 |
+----------------------------+----------------------+-----------------+
| *E. coli* DNA Ligase (10   | Invitrogen           | 18052-019       |
| U/ul, 100U)                |                      |                 |
+----------------------------+----------------------+-----------------+
| *E. coli* DNA polymerase I | Invitrogen           | 118010-017      |
| (10 U/ul, 250U)            |                      |                 |
+----------------------------+----------------------+-----------------+
| RNaseOUT RNase Inhibitor   | Invitrogen           | 10777-019       |
| (40u/ul, 5,000u)           |                      |                 |
+----------------------------+----------------------+-----------------+
| *E coli* RNaseH (2U/ul,    | Invitrogen           | 18021-014       |
| 30U)                       |                      |                 |
+----------------------------+----------------------+-----------------+
| T4 DNA polymerase (5U/ul,  | Invitrogen           | 18005-017,      |
| 50, 250U))                 |                      | 18005-025       |
+----------------------------+----------------------+-----------------+
| Glycogen (20ug/ul, 100ul)  | Invitrogen           | 18014-010       |
+----------------------------+----------------------+-----------------+
| dT~15~VN~2~ primer (50uM)  | IDT                  |                 |
+----------------------------+----------------------+-----------------+
| Phenol chloroform isoamyl  | Fluka                | 77617           |
| alcohol (PCI,25:24:1)      |                      |                 |
|                            | Sigma                | P4557           |
| Phenol in Tris             |                      |                 |
+----------------------------+----------------------+-----------------+
| Ultra Pure Ethidium        | Invitrogen           | 15585011        |
| Bromide (Carcinogen)       |                      |                 |
| (10mg/ml)                  |                      |                 |
+----------------------------+----------------------+-----------------+
| 50bp DNA ladder (0.5ug/ul, | MBI Fermentas        | SM0371          |
| 50ug)                      |                      |                 |
+----------------------------+----------------------+-----------------+
| 100bp DNA ladder Plus      | MBI Fermentas        | SM0323          |
| (0.5ug/ul, 50ug)           |                      |                 |
+----------------------------+----------------------+-----------------+
| Minelute Gel Extraction    | Qiagen               | 28604, 28606    |
| Kit (50,250)               |                      |                 |
+----------------------------+----------------------+-----------------+
| MinElute Reaction Cleanup  | Qiagen               | 28204, 28206    |
| Kit (50, 250)              |                      |                 |
+----------------------------+----------------------+-----------------+
| Buffer QG (250 ml)         | Qiagen               | 19063           |
+----------------------------+----------------------+-----------------+
| Buffer PE (concentrate,    | Qiagen               | 19065           |
| 100 ml)                    |                      |                 |
+----------------------------+----------------------+-----------------+
| ***                        |                      |                 |
| [Equipment]{.underline}*** |                      |                 |
+----------------------------+----------------------+-----------------+
| SpeedVac (optional)        |                      |                 |
+----------------------------+----------------------+-----------------+
| Agarose gel box and 1.5mm  | Owl                  | D-14            |
| 13 teeth combs             |                      |                 |
+----------------------------+----------------------+-----------------+
| NanoDrop spectrophotometer | NanoDrop             | ND1000          |
+----------------------------+----------------------+-----------------+
| Bioanalyzer                | Agilent              | 2100            |
+----------------------------+----------------------+-----------------+
| Micro centrifuge           | Eppendorf            | 5415-C          |
| (1000-16000RCF)            |                      |                 |
+----------------------------+----------------------+-----------------+
| Thermoblock                | Eppendorf            | 022670522       |
+----------------------------+----------------------+-----------------+
| Vortex                     | VWR                  |                 |
+----------------------------+----------------------+-----------------+
| Mini centrifuge            | VWR                  |                 |
+----------------------------+----------------------+-----------------+
| Promega Vac-Man Vacuum     | Fisher               | PR-A7231        |
| Manifold                   |                      |                 |
+----------------------------+----------------------+-----------------+

# Procedure -- cDNA Library construction 

All reagents/stock solutions should be prepared prior to the start of
the procedure. Once PolyA RNA is thawed to 4C, work quickly to prevent
RNA degradation. Wear gloves to protect samples from your hands and
protect your hand from harmful chemicals. Change gloves often. Put on a
new pair of gloves before handling critical samples. Change gloves after
handling tubes containing harmful agents such as phenol, Ethidium
Bromide (Carcinogen).

# cDNA Synthesis Protocol

The protocol provides the essential info needed for an experienced user.
Refer to the specific sections for important details.

1.  [First-strand cDNA synthesis]{.underline}, set up 2 tubes for 2ug
    polyA RNA, for each tube:

-   add the following components to a nuclease-free 0.2ml PCR tube:

> X ul Nuclease-free H~2~O
>
> 1-10ul 1ug polyA RNA
>
> 1ul dT~15~VN~2~ primer (50uM)
>
> 1ul 10mM dNTP
>
> \-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--
>
> 12ul Total

-   briefly mix, spin..

-   65C, 5min. 4C 2min.

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-   add the following components:

> 4ul 5x First-Strand buffer
>
> 1ul 0.1M DTT
>
> 1ul RNaseOUT (40u/ul)
>
> 2ul SuperScript III RT (200u/ul)
>
> \-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--
>
> 20ul Total

-   50C, 1h

-   70C, 15min

-   4C, 2 min, spin the content down

2.  [Second strand synthesis]{.underline}, for each tube:

-   add the following component:

> 91ul Nuclease-free H~2~O
>
> 30ul 5X Second-Strand buffer
>
> 3ul 10mM dNTP
>
> 1ul E coli DNA ligase(10U/ul)
>
> 4ul E coli DNA polymerase(10U/ul)
>
> 1ul E coli RNaseH (2 U/ul)
>
> \-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\-\--
>
> 150ul Total

-   briefly vortex mix, spin, remove content into 1.5ml Nuclease-free
    tube

-   16C, 2h

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-   put the tube on ice, add 2ul T4 DNA polymerase(5U/ul)

-   briefly vortex mix, spin

-   16C, 5 min

-   place the tube on ice

-   add 10ul 0.5M EDTA pH8, to stop the reaction (optional)

-   briefly vortex mix, spin

3.  [Phenol chloroform (Corrosive) isoamyl alcohol (PCI) and EtOH
    precipitation]{.underline}

> PPE NOTE: Extraction should be done in hood wearing safety goggles.
> Waste should be disposed of in a separate designated container.
>
> Important Safety NOTE: Phenol is a corrosive agent which burns skin
> upon contact, practice extreme safety when performing the following
> steps**.**

-   spin two Eppendorf phase-lock Light tubes (Green)

-   remove cDNA reaction content to phase-lock tube, transfer tubes to
    fume-hood. (lab#459)

-   add 150ul PCI/tube, mix by shaking the tube vigorously for 15 second

-   centrifuge @ 13000g RT, 5min

-   transfer top layer into 1.5ml clean Nuclease-free tube

-   for each tube, add the following component:

> 15ul (1/10vol) 3M NaOAc, PH5.2
>
> 1.2ul Glycogen (to help precipitate DNA)
>
> 415ul (2.5vol) EtOH

-   mix by inverting the tubes.

-   place the tubes in -70C for \>1h (optional).

-   centrifuge @ 13000g RT for 20min. (prepare 2% agarose gel while
    waiting).

-   remove tubes from centrifuge gently, look for a white \~1 mm size
    pellet on the hinge side. This pellet should be present even if
    there were no DNA/RNA. If not present, check to see if all reagents
    were added. Spin again if needed to see the pellet.

-   remove the supernatant with tip pointing away from the pellet, check
    to make sure the pellet remains after you remove the supernatant

-   wash pellet with 500ul 70% EtOH

-   centrifuge @ 13000g RT for 2min.

-   remove the supernatant, air dry \@RT for 10min or SpeedVac dry for
    5min

-   resuspend pellets of 2 tubes in total 15ul of nuclease-free H~2~O
    (sample can be stored @ -20C).

4.  [Gel separation]{.underline}

-   prepare 2% 1XTAE agarose gel 0.1 mg/ml Ethidium Bromide (Carcinogen)
    (need \~30min)

-   save 2ul of sample *(as a control)* and run gel-purified sample
    together on Bioanalyzer

-   add 2.5ul 5x loading dye (Qiagen) to remaining sample

-   pipette mix and load on gel, load 50bp & 100bp DNA ladder
    (Fermentas)

-   run gel @ 100V for \~10 min *(to let the sample migrate from well in
    to gel quickly)* then \@50V for \~30min (*cm to maximize separation
    of small molecular weight primer away from the cDNA)*

-   image gel for 1 second *(to minimize UV exposure)* and save photos
    of both pre-cut and post-cut gels. cDNA are normally not visible \@1
    second exposure time, gel picture can be adjusted later so that cDNA
    are visible. The primer should run below 50bp.

-   cut gel slice from 100bp up to well using UV transluminator *(make
    sure not to include the primer into your gel piece)*, place it in a
    15 or 50ml falcon tube

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-   *Important! Minimize UV exposure to yourself (use long sleeves lab
    coats and face shield) and to the samples (use laminated paper to
    block UV to the samples not being cut). Turn off UV after cutting
    each sample. Transfer agar pieces to tubes with normal light. Review
    cut gel to make sure the right pieces of gel were transferred.*

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-   take another gel picture with 5 second exposure time.

-   clean the gel and image area (gel slice can be stored @-20C)

5.  [Gel extraction]{.underline}

-   weight the gel slice

-   add 3X volume of QC buffer

-   rotate the tube \@37C or 50C incubator for 15 or 10min respectively,
    until no visible gel slice present

-   add 1X volume of isopropanol, mix

-   the following steps can be performed by vacuum or centrifuge
    (13000g, 1min per)

a.  load the sample to Minelute column

b.  applied the column to vacuum manifold or centrifuge, repeat this
    step 3-4 times if you have more than 750ul (load sample to another
    column if you have more than 3ml)

c.  apply 500ul QG buffer for each column, vacuum or centrifuge

d.  apply 750ul PE buffer for each column, vacuum or centrifuge, repeat
    this step once

-   place the column into emptied 2ml tube, centrifuge \@13000g, 1min

-   rotate tube 180C, centrifuge \@13000g, 30second

-   place the column to a nuclease-free 1.5ml tube

-   add 12ul EB, wait 1min

-   centrifuge \@13000g, 1min.

-   use same EB solution to elute the other tube of the same sample
    (sample can then be stored @ -20C)

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```
-   determine cDNA concentration by Bioanalyzer (Bioanalyzer 7500 kit).

-   cDNA should be \>80ng/ul (total \>1ug) if you start with 2ug PolyA
    RNA.

6.  Sonication

-   use pump to circulate ice water to cool sonicator to 4C.

-   dilute \>1ug cDNA in 100ul TE in a 1.5 microfuge tube.

-   place tube in a rack in water bath that allows the bottom of the
    tube to touch the sonicator horn.

-   make sure the liquid level of the sample is below the sonicator
    bath.

-   sonicate for 8 min at 1 min intervals with 1 min off between to
    allow sample to cool.

-   spin 10sec.

-   Minelute purify cDNA, elute in 12ul

-   determine DNA concentration and size by Bioanalyzer DNA1000 kit.

-   cDNA should be \>80ng/ul (total \>1ug) range 100-800bp.

-   gel purify cDNA between 100 to 800bp if needed (see step 4, 5 for
    details)

7.  GS20 DNA library preparation

-   End Polishing and Purification

-   Adaptor ligation

-   Melt DNA to isolate single stranded DNA

-   Use picoRNA kit estimate ssDNA concentration.

-   Emulsion PCR with library beads.

-   Load beads to 454 plates.

# References

+-----------------------+-----------------------+-----------------------+
| **Source**            | **Authors**           | **DATE**              |
+-----------------------+-----------------------+-----------------------+
| cDNA Library          | Dean Ng, etal         | 3/12/07               |
| Construction SOP 4.1  |                       |                       |
+-----------------------+-----------------------+-----------------------+
| GS20 DNA Library      | ######## Jamie Jett   | 10/9/06               |
| Preparation           |                       |                       |
+-----------------------+-----------------------+-----------------------+
| [Agilent DNA 7500 and | ########              |                       |
| DNA 12000 Kit Quick   |  Agilent Technologies |                       |
| Start                 |                       |                       |
| Guide](ht             |                       |                       |
| tp://www.genomics.bha |                       |                       |
| m.ac.uk/DNA_Kits.pdf) |                       |                       |
+-----------------------+-----------------------+-----------------------+
| [SuperScript Plasmid  | ######## Invitrogen   |                       |
| System with Gateway   |                       |                       |
| Technology for cDNA   |                       |                       |
| Synthesis and         |                       |                       |
| Cl                    |                       |                       |
| oning](http://www.inv |                       |                       |
| itrogen.com/content/s |                       |                       |
| fs/manuals/18248.pdf) |                       |                       |
+-----------------------+-----------------------+-----------------------+
| [SuperScriptIII       | ######## Invitrogen   |                       |
| Reverse               |                       |                       |
| Transcriptase](http:/ |                       |                       |
| /www.invitrogen.com/c |                       |                       |
| ontent/sfs/manuals/su |                       |                       |
| perscriptIII_man.pdf) |                       |                       |
+-----------------------+-----------------------+-----------------------+
| [MinElute Gel         | ######## Qiagen       |                       |
| Extraction            |                       |                       |
| Kit](http:            |                       |                       |
| //www1.qiagen.com/lit |                       |                       |
| erature/handbooks/PDF |                       |                       |
| /DNACleanupAndConcent |                       |                       |
| ration/MinElute/10360 |                       |                       |
| 20_Qiag_Complete.pdf) |                       |                       |
+-----------------------+-----------------------+-----------------------+

# Oligonucleotides

dT~15~VN~2~ : TTTTTTTTTTTTTTTVNN

# 5X first strand buffer

250mM Tris HCl pH8.3, 375mM KCl, 15 mM MgCl~2~

# 5X Second Strand buffer

100mM Tris HCl pH6.9, 450mM KCl, 23 mM MgCl~2~, 0.75mM β-NAD^+^, 50mM
(NH~4~)~2~SO~4~

# SOP Approval

  ----------------------- ----------------------- -----------------------
  DEPARTMENT              APPROVED BY             DATE

  Lab Supervisor          Feng Chen               

  Research and            Feng Chen               
  Development                                     

  Instrumentation                                 

  QC                                              

  Purchasing                                      

  EH and S                                        

  Dept Head of Prod Seq   Paul Richardson         
  ----------------------- ----------------------- -----------------------