Supplemental methods
Preparation of size-fractionated npcRNA
Total RNA was isolated from cultures containing all developmental stages of C. elegans
(strain Bristol N2) by the TRIzol method (Invitrogen Life Technologies). 200 µg of total RNA
was fractionated on a denaturing 8% acrylamide/bisacrylamide (19:1; 7M Urea) gel. RNAs in the
size range of 70-600 nt were excised and eluted from the gel (Hüttenhofer et al., 2001).
cDNA synthesis and cloning (SuperScript technology)
Briefly, 5 µg of size-fractionated RNA was tailed with CTP using poly(A) polymerase.
First strand synthesis (reverse transcription) was primed with an oligonucleotide containing
oligo(G)15 and a NotI restriction site for subsequent cloning. Second strand synthesis used the
first strand cDNA as a template and substituted the RNA by nick translational replacement
according to the manufacturer’s instructions (SuperScript system, Invitrogen). A double stranded
SalI linker adapter was ligated to the 5’ end of the cDNA providing a precut SalI restriction site.
After digestion with NotI and elution from a non-denaturing 6% acrylamide/bisacrylamide (19:1)
gel, the cDNA was ligated into a SalI/NotI-digested pSPORT 1 vector (Invitrogen) and
transformed into E. coli TOP10 cells (Invitrogen).
High-density arrays
About 200 cDNAs were initially sequenced to check the library quality and to obtain a
survey of the most abundant known npcRNAs. Based on this information we generated 33P-ATPlabeled oligonucleotides complementary to, for example rRNAs, known small npcRNAs, or
vectors without inserts. To avoid known npcRNAs we amplified the cDNA library with M13
primers and robotically spotted the PCR fragments on high-density arrays (Schmitt et al., 1999).
The arrays were then hybridized with the labeled oligonucleotides for 12 h in 0.5 M sodium
phosphate (pH 7.2), 7% SDS, 1 mM EDTA at 53°C. After washing twice at room temperature for
15 min (40 mM sodium phosphate pH 7.2, 0.1% SDS) the filters were exposed to a
phosphorimaging screen. Strong hybridization signals, indicating known RNA sequences or
vector contaminations, were excluded from further sequencing (Hüttenhofer et al., 2001). The
efficiency of pre-screening and exclusion of known npcRNAs is illustrated in supplementary
Figure S-1 online.
Sequencing cDNA clones
PCR fragments of the cDNA clones were generated with M13 primers. Sequencing was
performed using the Ampli Taq FS Big Dye Terminator Kit (PE Biosystems, Foster City, Calif.)
and M13 primers (Supplementary materials).
Northern blot hybridization
Total RNA was separated on 8% denaturing acrylamide/bisacrylamide (19:1; 7M Urea)
gels, transferred onto nylon membranes (BrightStar-Plus, Ambion), UV- crosslinked (Stratagene
crosslinker) and pre-hybridized for 1 h at 58°C in 1M sodium phosphate buffer (pH 6.2), 7%
SDS. Hybridizations to
32
P-ATP, end-labeled oligonucleotides complementary to the respective
npcRNAs (supplemental Table 1 and 2 online) were performed in 1 M sodium phosphate (pH
6.2), 7% SDS for 12 h at 58°C. Membranes were washed twice for 15 min at room temperature in
2x SSC, 0.1% SDS buffer, twice for 15 min at room temperature in 0.1x SSC, 0.1% SDS, and
exposed to Kodak MS-1 films for 3 h to 2 days.
Pattern Search of DNAMAN (provided by the Lynnon Corporation, Canada)
The algorithm is described in brief followed with a psudocode.
1. Pattern decomposition
Patterns allow various ambiguities, matches to variable lengths of sequence and repeated
subsections of the sequence. In this step, the pattern is decomposited to motifs, motif
intervals and mismatches. The three components are individually stored in arrays.
2. Motif match module
This module defines the method of comparison of motifs to target sequence. It is called
repetitively in pattern searching process.
3. Search pattern using dynamic programming
In this step, motifs are searched recursively according to the order. If every motif is found
in a searching run, the pattern is complet and the position is recorded.
Psudocode
Algorithm
Pattern_Search
Input
pattern P, sequence S
Output
list of all occurrences of P in S
Begin
/* Pattern Decomposition */
Extract P to
motif list MA
motif interval list IA
mismatch list EA
/* End Pattern Decomposition */
/* Do recursive search */
Do_Recursive_Search (MA, IA, EA)
Return occurrence_list
End
Algorithm
Input
Output
Begin
Do_Recursive_Search
sequence S, motif list MA, motif interval list IA, mismatch list EA
list of MA positions in S
If (all MA searched) then record S position in the list of occurrences
Else /* continue searching */
found_list = Motif_Match (next MA, S, next IA, next EA)
For (1, number of found_list)
Do_Recursive_Search (MA, IA, EA)
Return
End
Algorithm
Input
Output
Begin
Motif_Match
motif M, sequence S, mismatch allowed E
list of match positions
Define searching positions in S: start at P1, end at P2
For (P1, P2)
mismatch = Compare M to S at each position
If (mismatch < E) then record the position in list
Else reset mismatch and continue
Return
End
Literature
Hüttenhofer, A., Kiefmann, M., Meier-Ewert, S., O'Brien, J., Lehrach, H., Bachellerie, J.-P. and
Brosius J. 2001. RNomics: an experimental approach that identifies 201 candidates for
novel, small, non-messenger RNAs in mouse. EMBO 20: 2943-2953.
Schmitt, A.O., Herwig, R., Meier-Ewert, S. and Lehrach, H. 1999. High density cDNA grids for
hybridization fingerprinting experiments. In Innis, M.A., Gelfand, D.H. and Sninsky, J.J.
(eds), PCR Applications: Protocols for Functional Genomics. Academic Press, San
Diego, CA, pp. 457-472.