rRNA sequencing using reverse transcriptase

　Sequencing of rRNAs, primarily 16S rRNA at this time, is a useful method for quickly determining the phlyogenetic placement of an organism. Because of the presence of highly conserved regions in the 16S rRNA, universal oligonucleotide primers are available for sequencing portions of the 16S rRNAs from about any organism. These primers are situated in downstream of regions that are quite variable; these are sequences which are quite useful for phylogenetic analysis. The method described here is, of course, also aplicable for 23S rRNA sequencing provided appropriate oligonucleotide primers are used. The method can even be used for sequencing abundant mRNAs - in this case, it might be useful to modify the method by using end-labeled primers rather than incorporating radiolabeled dNTPs into the reaction mixes.
　The most important part of this procedure is the RNA isolation. Use RNase-free technique! Otherwise, the procedure is not essentially different from chain termination sequencing from DNA templates.
　If the sequences are difficult to interprete, try re-precipitating the RNA in 2M NaCl (0C overnight) to remove low MW RNAs. Also try decreasing the primer concentration in the sequencing reactions 5-fold (i.e. use an 0.02mg/ml stock concentration).

Reagents
1. 5X hybridization buffer
  
  100mM KCl
  
  250mM Tris, pH8.5
2. 5X RT buffer
  
  250mM Tris, pH8.5
  
  50mM DTT
  
  50mM MgCl2
3. RT dilution buffer
  
  50mM Tris, pH8.3
  
  2mM DTT
  
  50% glycerol
4. Sample buffer
  
  86% formamide
  
  10mM EDTA
  
  0.08% xylene cyanol
  
  0.08% bromophenol blue
5. Gel fix - 10% methanol, 10% glacial acetic acid, 2% glycerol
6. Oligonucleotide primers - dissolved in ddH2O to 0.1mg/ml
  primer 519 5'-GWAATACCGCGGCKGCTG-3'
  primer 915 5'-GCCCCCGYCAATTCCT-3'
  primer 1391 5'-GACGGGCGGTGTGTRCA-3'

PREPARATION OF NUCLEOTIDE MIXES

For dNTPs, dissolve 10mg in 1ml 10mM Tris, pH8.3. For ddNTPs, dissolve 4umoles in 300ul 10mM Tris, pH8.3
Dilute 4ul of this into 2ml 10mM Tris, pH 7.4, and read the absorbance at that (d)dNTPs absorption maximum - the (d)dNTP concentration can be calculated as:

(absorbance)(dilution factor)/(mM extintion coefficient) = concentration in mM

The constants needed are:

nucleotide	absorption max (nm)	mM extinction coeff
dATP	259	15.2
dCTP	272	9.1
dGTP	253	13.7
dTTP	267	9.6
dITP	249	12.2
[S]dATP	259	14.8
ddATP	259	15.2
ddCTP	272	9.1
ddGTP	253	13.7
ddTTP	267	9.6

Dilute the initial solution to 10mM by adding 10mM Tris, pH8.3.

To the alpha-thio-dATP solution, add 1/1000 volume (1ul/ml) 1M DTT.
Store at -70C.
Make small (~500ul) working stocks of each of the following in 10mM Tris, pH8.3: 1mM ddCTP, 1mM ddGTP, 1mM ddTTP, and 0.1mM ddATP.

Six nucleotide mixes are needed for RT sequencing (numbers are in ul):

	G-mix	C-mix	T-mix	A-mix	no-dd mix	chase
10mM dGTP	10	10	10	10	10	140
10mM dCTP	10	10	10	10	10	140
10mM dTTP	10	10	10	10	10	140
10mM [S]dATP	5	5	5	5	5	-
10mM dATP	-	-	-	-	-	140
1mM ddGTP	7.6	-	-	-	-	-
1mM ddCTP	-	12	-	-	-	-
1mM ddTTP	-	-	12	-	-	-
0.1mM ddATP	-	-	-	5	-	-
10mM Tris,pH8.3	353	349	349	356	361	833
1M MgCl2	-	-	-	-	-	7
100mM DTT	4	4	4	4	4	-

Divide the mixes into 20ul aliquots, except chase mix, which gets 70ul aliquots, and store at -20C.

RNA PREPARATION

　RNA can be prepared by standard methods, depending on the cell type. High molecular weight RNA should be removed from total RNA preparations by NaCl precipitation, or rRNA should be isolated by phenol extraction & ethanol precipitation from ribosomal pellets (i.e. S100 pellets). rRNA should be dissolved in ddH2O at a concentration of about 2mg/ml.

REVERSE TRANSCRIPTASE SEQUENCING REACTIONS

　The sequencing reactions are easiest done in sets of three primers (i.e. 3 sets od sequencing reactions). This allows you to use the standard three universal primers quickly.

Prepare the following tubes:

Hybrid mix	A	B	C
5X hybridization buffer	1.4ul	1.4ul	1.4ul
template RNA (2mg/ml)	4.0ul	4.0ul	4.0ul
primer A (0.1mg/ml)	1.5ul	-	-
primer B (0.1mg/ml)	-	1.5ul	-
primer C (0.1mg/ml)	-	-	1.5ul

Incubate at 65C for 2 min., then slow-cool to room temperature over about 10min., then prepare the following tubes, on ice:

Pre-mixes	A	B	C
[alpha-35S]dATP (30uCi)	dry	dry	dry
5X RT buffer	6.0ul	6.0ul	6.0ul
Hybrid mix A	6.0ul	-	-
Hybrid mix B	-	6.0ul	-
Hybrid mix C	-	-	6.0ul
1000units/ml RT	6.0ul	6.0ul	6.0ul

The RT is diluted to 1u/ul (1000u/ml) in RT dil'n buffer just prior to addition.

Still working on ice, prepare the following mixes for each premix (in is best to have the dNTP/ddNTP mixes pre-aliquoted into tubes, ready for the addition of the pre-mixes):

Sequencing reactions		A	B	C
A	pre-mix	3.0ul	3.0ul	3.0ul
	A-mix	2.0ul	2.0ul	2.0ul
G	pre-mix	3.0ul	3.0ul	3.0ul
	A-mix	2.0ul	2.0ul	2.0ul
C	pre-mix	3.0ul	3.0ul	3.0ul
	A-mix	2.0ul	2.0ul	2.0ul
T	pre-mix	3.0ul	3.0ul	3.0ul
	A-mix	2.0ul	2.0ul	2.0ul
Control	pre-mix	3.0ul	3.0ul	3.0ul
	no dd mix	2.0ul	2.0ul	2.0ul

Incubate at room temperature for 5 min., then 30 min. at 55C.
Add 1ul chase mix (10:1 chase mix:1000u/ml RT) & incubate 15 min., 55C.
Dry the samples in a rotovac, and redissolve each sample in 10ul sample buffer. Electrophorese on 6% sequencing gels: two loadings are best, with the second started as the first samples xylene cyanol reaches 2/3rds of the way to the bottom of the gel. Run the gel until the bromophenol blue of the seconf loading reaches the bottom of the gel. Soak the gel in Gel soak for 15 min., then dry and autoradiograph. Try a 2 day exposure.

100mM	KCl
250mM	Tris, pH8.5

250mM	Tris, pH8.5
50mM	DTT
50mM	MgCl2

50mM	Tris, pH8.3
2mM	DTT
50%	glycerol

86%	formamide
10mM	EDTA
0.08%	xylene cyanol
0.08%	bromophenol blue