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LyX: Attempting to save document
/home/maasha/promoter_paper/promoter_paper_0.10.lyx
/home/maasha/promoter_paper/promoter_paper_0.10.lyx.emergency
Save seems successful. Phew.
this happens, when i try to merge changebars.
the error lies within this:
Bioinformatics
\layout Standard
The human genome sequence used for all BLAST searches was the NCBI Build
35 version produced by the International Human Genome Sequencing
Consortium.
\layout Standard
Three cycles of iterative BLAST were performed using the murine CYPT
promoter
sequence
\begin_inset LatexCommand \citep{Hansen2005}
\end_inset
as the initial query sequence and the human genome as subject sequence.
For each cycle the results was filtered using a custom P
\change_inserted 1 1133882579
erl
\change_deleted 1 1133882579
ERL
\change_unchanged
script to remove overlapping subject sequences except the longest, and
the next cycle was performed using the previous query sequences and the
previous non-overlapping subject sequences as the new query sequences.
The search was performed using WU-BLAST
\begin_inset LatexCommand \cite{Gish1996-2004}
\end_inset
with the following parameters: filter=none, hspmax=0, hspsepQmax=500,
hspsepSma
x=1000, and E=0.0001.
Sequence alignment was performed with Muscle (default parameters)
\begin_inset LatexCommand \cite{Edgar2004}
\end_inset
.
Dotplots were generated with a custom P
\change_inserted 1 1133882579
erl
\change_deleted 1 1133882579
ERL
\change_unchanged
script plotting the output of NCBI bl2seq megaBLAST (default parameters)
alongside annotations fetched from the Ensembl application program
interface
(API).
\layout Subsection*
Preparation of Biotin Labeled Probes for
\emph on
In\SpecialChar ~
Situ
\emph default
Hybridization
\begin_inset Note
collapsed true
\layout Standard
cDNA syntese ikke beskrevet - ref Jorgensen2000
\end_inset
\layout Standard
\emph on
In\SpecialChar ~
situ
\emph default
hybridization (ISH) probes were designed for the human genes and ESTs found
downstream of the CYPT promoter.
The ISH probes were prepared using a two-step PCR approach, where the first
PCR round for each target gene family or EST (Table\SpecialChar ~
\begin_inset LatexCommand \ref{Table: primers}
\end_inset
; column\SpecialChar ~
1) was run with the forward- (Table\SpecialChar ~
\begin_inset LatexCommand \ref{Table: primers}
\end_inset
; column\SpecialChar ~
2) and reverse primers (Table\SpecialChar ~
\begin_inset LatexCommand \ref{Table: primers}
\end_inset
; column\SpecialChar ~
3).
The second PCR round was run with T3- (Table\SpecialChar ~
\begin_inset LatexCommand \ref{Table: primers}
\end_inset
; column\SpecialChar ~
4) and T7- (Table\SpecialChar ~
\begin_inset LatexCommand \ref{Table: primers}
\end_inset
; column\SpecialChar ~
5) tagged primers on 1
\begin_inset Formula $\mu$
\end_inset
l of the crude product from the first PCR round.
\change_deleted 1 1133882579
\change_unchanged
\layout Standard
PCR conditions were: first PCR round: 5\SpecialChar ~
min at 95°C; 30\SpecialChar ~
cycles of: 30\SpecialChar ~
sec at
95°C, 1\SpecialChar ~
min at 62°C, 1\SpecialChar ~
min at 72°C; and finally 5\SpecialChar ~
min at 72°C.
1
\begin_inset Formula $\mu$
\end_inset
l (out of 30
\begin_inset Formula $\mu$
\end_inset
l total volume) was transferred to a new PCR reaction and run as follows:
5\SpecialChar ~
min at 95°C; 5\SpecialChar ~
cycles of: 30\SpecialChar ~
sec at 95°C, 1\SpecialChar ~
min at 45°C, 1\SpecialChar ~
min at 72°C; 30\SpecialChar ~
cycles
of 30\SpecialChar ~
sec at 95°C, 2\SpecialChar ~
min at 72°C and finally 5\SpecialChar ~
min at 72°C.
The resulting PCR product were purified on 2% low-melting agarose gels
and sequenced from both ends, using Cy5-labelled primers complementary
to the added T3- and T7-promoter tags.
Aliquots of 200ng were used for
\shape italic
in\SpecialChar ~
vitro
\shape default
transcription labelling, using the MEGAscript-T3 (sense) or MEGAscript-T7
(anti-sense) kits, as described by the manufacturer (Ambion, Houston, TX,
USA).
The composition of the 10x-nucleotide mix was: 7.5mM ATP, GTP, and CTP
along
with 3.75mM UTP and 1.5mM biotin-labelled UTP.
The labeled probes were subsequently used for ISH as previously described
\begin_inset LatexCommand \cite{Nielsen2003}
\end_inset
.
\layout Standard
ideas?
martin
