Supplementary Methods
MATERIALS
REAGENTS
ELONGASE enzyme mix (Invitrogen)
ExTaq DNA polymerase (Takara)
Ligation Kit Version 2 (Takara)
Proteinase K (QIAGEN)
pZErO-2 plasmid DNA (Invitrogen)
Restriction enzymes MmeI (New England Biolabs) and XmaJI (Fermentas)
RNase I (Promega)
SuperScript II RNase H reverse transcriptase (Invitrogen)
T4 DNA Ligase (New England Biolabs)
tRNA (transfer RNA) type XX from Escherichia coli, strain W (Sigma)
EQUIPMENT
Magnetic particle concentrator (Dynal)
Filtrate assembly: 96-place holder & 96-well receiver plate (Microcon-96, Millipore)
Microcon YM-100 columns, centrifugal filter unit and 96-well retentate assembly plate
(Millipore)
MicroSpin columns (empty, G-50, S-400; Amersham Biosciences)
Open tip mini column (E&K Scientific)
QIAvac 96: vacuum manifold for processing QIAGEN 96-well plates including QIAvac
96 top plate, base, waste tray, plate holder and rack for collection microtubes (1.2 ml)
(QIAGEN)
Thermal cycler, programmed with desired amplification protocol
Thermo-Fast 96-well plate (ABgene)
UNIFILTER GF/C 800 filter microplate (Whatman)
Protocol 1 Reverse transcription using oligo(dT) primers
Standard oligo(dT) primers can be used in place of random primers for reverse
transcription, in particular if a parallel preparation of a cDNA library from the same
starting material is planned. Instructions for reverse transcription using oligo(dT) are
given below and may be used as an alternative to Steps 1–4. Resume the main protocol at
Step 5.
1. Precipitate and recover total RNA as described in Step 1 of the main protocol;
resuspend the pellet in 15 µl of water.
2. Transfer the RNA solution to 96-well PCR microtiter Plate A and add 7 µl (2 µg/µl) of
oligo(dT) primer.
3. Prepare the reaction mix in Plate B as described in Step 3.
4. Heat Plate A to 65 °C for 10 min and then place immediately on ice. Warm Plates A
and B at 42 °C for 1.5 min and then transfer the contents from Plate B to Plate A. Carry
out reverse transcription in a thermal cycler as follows: 30 min at 42 °C, 10 min at 50 °C,
10 min at 56 °C; hold at 4 °C until further processing. Continue with Step 5 of the main
protocol.
Protocol 2 Fractionation of the ligation mixture using Sephacryl S-400 resin
Passage of the ligation mixture over a Sephacryl S-400 column removes small
contamination nucleic acids, including residual linkers and linker-dimers. The following
protocol describes purification of the ligation mixture in Step 32 of the main protocol.
1. Add 2 ml Sephacryl S-400 HR resin to open tip mini columns and equilibrate with
CL4B buffer according to the manufacturer’s directions.
2. Test the column by applying 100 µl CL4B buffer, ensuring that ~100 µl CL4B buffer
can be recovered after centrifugation. If not, repeat equilibration with another 100 µl
CL4B buffer.
3. When ~100 µl solution has been recovered, proceed with the application of the first
cDNA extract (60 µl) to the column followed by the second (40 µl) (Step 31).
4. Centrifuge the column at 100g in a swing-out rotor for 2 min at 15–25 °C and then
transfer the eluate to a fresh tube. Start collecting the following fractions into the same
tube.
5. Apply 50 µl CL4B buffer to the middle of column and centrifuge again as in Step 1.
6. Repeat Step 5 twice.
7. Recover the DNA by precipitation with isopropanol under standard conditions.
Protocol 3 Elution of DNA from polyacrylamide
DNA is eluted from the polyacrylamide gel using a salt buffer. The following protocol
describes the recovery of DNA from the gel in Step 54 of the main protocol.
1. Crush the gel pieces in a 1.5 ml microcentrifuge tube, add 150 µl of PAGE elution
buffer and elute at 15–25 °C overnight.
2. Incubate the tube at 65 °C for 30 min.
3. To separate the DNA from the acrylamide, transfer the supernatant to the empty
MicroSpin columns and centrifuge at 735g at 15–25 °C for 2 min.
4. Repeat the elution step twice, each time adding 150 µl PAGE elution buffer and
incubating at 65 °C for 30 min.
5. To recover the remaining DNA, perform back extraction using 50 µl LoTE without
incubation. Collect (pool) the fractions (of the various elution steps) in one vial.
Protocol 4 CAGE tag analysis
CAGE tags can be extracted from sequencing reads using the PERL scripts provided on
our homepage at http://genome.gsc.riken.jp/CAGE and mapped to genomic sequences
under standard conditions1. Note that the PERL scripts will work properly only if you use
the same linker sequences as given in this protocol!
For CAGE tag mapping to the mouse or human genome2, we used BLAST3 under default
settings, but the penalty for a nucleotide mismatch was set to –1 (parameter q = –1).
Successfully mapped tags have a minimum of 18 bp continuous homology to genomic
sequences. For longer tags of 21 bp or more, a gap of 1 mismatch in the center of the tag
was permitted.
REFERENCES
1.
Shiraki, T. et al. Cap analysis gene expression for high-throughput analysis of
transcriptional starting point and identification of promoter usage. Proc. Natl.
Acad. Sci. USA 100, 15776–15781 (2003).
2.
Kawaji, H. et al. CAGE Basic/Analysis Databases: the CAGE resource for
comprehensive promoter analysis. Nucl. Acids Res. 34 Database issue), D632–
D636 (2006)
3.
Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. Basic local
alignment search tool. J. Mol. Biol. 215, 403–410 (1990).