1. Digital Gene Expression – Tag Profiling 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 1-2ug of purified total RNA

2. Digital Gene Expression – Tag Profiling Sample Preparation
2. Isolate mRNA and Synthesize First Strand cDNA
mRNA is isolated from total RNA by binding the mRNA to a magnetic oligo(dT) bead.
mRNA has a polyA tail and will bind to the oligo(dT) bead.
Using the mRNA attached to the bead as a template, oligo(dT) bound cDNA is
synthesized to form a bead-bound mRNA/cDNA hybrid.
mRNA
5’-AGUGAUCAGGGUUCAAUAAAAAAAAAAAAA-3’
5’-AGUGAUCAGGGUUCAAUAAAAAAAAAAAAA-3’
PolyA Tail
Magnetic
Oligo (dT)
bead
Total RNA containing
mRNA is added to
magnetic oligo (dT)
beads
3’-TTTTTTTTTTTTT
3’-TTTTTTTTTTTTT
3’-TTTTTTTTTTTTT
5’-AGUGAUCAGGGUUCAAUAAAAAAAAAAAAA-3’
3’-TTTTTTTTTTTTT
First strand synthesis
5’-AGUGAUCAGGGUUCAAUAAAAAAAAAAAAA-3’
3’-TCACTAGTCCCAAGTTATTTTTTTTTTTTT

3. Digital Gene Expression – Tag Profiling Sample Preparation
2. Synthesize the Second Strand cDNA
Remove the strand of mRNA and synthesize a replacement strand generating double-stranded
cDNA bound to the oligo(dT) bead.
RNase H
5’-AGUGAUCAGGGUUCAAUAAAAAAAAAAAAA-3’
mRNA strand
3’-TCACTAGTCCCAAGTTATTTTTTTTTTTTT
cDNA strand
RNase H activity enzymatically degrades the mRNA strand
3’-TCACTAGTCCCAAGTTATTTTTTTTTTTTT
DNA Polymerase I activity generates second strand cDNA
to form double-stranded cDNA bound to oligo(dT) bead
DNA Polymerase I
5’-AGTGATCAGGGTTCAATAAAAAAAAAAAAA
3’-TCACTAGTCCCAAGTTATTTTTTTTTTTTT

4. Digital Gene Expression – Tag Profiling Sample Preparation
4. Restriction Digest with DpnII or NlaIII
Enzymatically cleave the double-stranded cDNA at every DpnII or NlaIII restriction site.
There is two digital gene expression kits available, one contains the restriction enzyme
DpnII and the other contains the restriction enzyme NlaIII. The customer has to decide which
kit to use.
DpnII restriction enzyme
5’-NGATCN-3’
3’-NCTAGN-5’
5’-AGTGATCAGGGTTCAATAAAAAAAAAAAAA
3’-TCACTAGTCCCAAGTTATTTTTTTTTTTTT
DpnII restriction enzyme cutting site
Restriction enzyme digestion
5’-NCATGN-3’
3’-NGTACN-5’
5’-GATCAGGGTTCAATAAAAAAAAAAAAA
3’-TCCCAAGTTATTTTTTTTTTTTT
NlaIII restriction enzyme cutting site
All fragments other than the 3’ fragment attached to the oligo(dT) bead are washed away.
NOTE: DpnII restriction enzyme cleavage is demonstrated

5. Digital Gene Expression – Tag Profiling Sample Preparation
5. Ligate GEX DpnII or NlaIII Adapter 1
A defined gene expression adapter (GEX DpnII or NlaIII Adapter 1) is ligated at the site of enzymatic
cleavage. The GEX DpnII or NlaIII Adapter 1 contains the sequence for the restriction enzyme MmeI,
Which is necessary for future steps in the sample preparation.
GEX DpnII adapter 5’ 3’ 3’
5’-GATCAGGGCGTACTTAAGTAGCTATTCAATCAGCAAAAAAAAAAAAA 5’
3’-TCCCGCATGAATTCATCGATAAGTTAGTCGTTTTTTTTTTTTT
GEX adapter binds at DpnII cleavage site
NOTE: DpnII adapter ligation is demonstrated

6. Digital Gene Expression – Tag Profiling Sample Preparation
6. Restriction Digest with MmeI
5’-TCCRACNNNNNNNNNNNNNNNNNNNNN-3’
3’-AGGRTGNNNNNNNNNNNNNNNNNNNNN-5’P
Restriction enzyme MmeI binding site
Restriction enzyme MmeI cutting site is 18bp
downstream from the enzymes binding site
This protocol applies the restriction enzyme MmeI to create the 16bp tag. The binding site for the enzyme is at the GEX DpnII/or GEX NlaIII/cDNA junction. The resulting construct is no longer attached to the oligo(dT) bead and is free in solution.
TTCAATCAGCAAAAAAAAAAAAA
AAGTTAGTCGTTTTTTTTTTTTT
Remove product from the magnetic beads
Into separate tube
Beads are left in original tube
5’-NNNNNNNNNNNNNNNNTCCRACNNNNNNNNNNNNNNNNNNNNN-3’
3’-NNNNNNNNNNNNNNNNAGGRTGNNNNNNNNNNNNNNNNNNNNN-5’P
Restriction enzyme MmeI binding site
Restriction enzyme MmeI cutting site

7. Digital Gene Expression – Tag Profiling Sample Preparation
7. Ligate GAX Adapter 2
This protocol ligates a defined gene expression adapter (GEX adapter 2) at the site of MmeI cleavage.
The GEX Adapter 2 contains sequences complementary to the oligo attached to the flow cell surface. 5’ 3’ 3’ 5’
MmeI cleavage site
GEX Adapter 2 5’ 3’ 3’ 5’
GEX Adapter 1
GEX Adapter 2
Product with GEX Adapter 1 ligated onto one end & GEX Adapter 2
ligated onto the other end.

8. Digital Gene Expression – Tag Profiling Sample Preparation
8. Enrich the Adapter-Ligated cDNA Construct using PCR
This protocol uses PCR to selectively enrich the DNA library with cDNA fragments
that have adapter molecules on both ends. The PCR is performed with two primers
that anneal to the ends of the adapters. 5’ 3’ 3’ 5’
Amplify products by PCR

9. Digital Gene Expression – Tag Profiling Sample Preparation
9. 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 85bp size range.
25bp ladder is in 25bp
Steps up to 300bp
100bp
8. Validate the Library
85bp
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 ng 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 ng of DNA.
Measure the OD 260/280 ratio, which should be ~1.8.
Sample
Ladder
Cut from gel
and purify