- Aug 2016
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Local file Local file
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potential
potentially
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s G
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Odds-Ratio
the odds ratio
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BiQAnalyzer compares the sequences in all aligned Cs and highlightsthe sequence as critical if they are identical in all aligned Cs. All these sequences were excluded in theanalysis.
This works because of incomplete conversion (<100%), plus actual differences in CpG methylation states.
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s
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n a
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Bonferroni correctionwas conducteddue to multiple comparisonsignificant valuelimitwas set to<0.025.
Bonferroni multiple testing correction: you divide alpha (the significance threshold), typically (by convention) 0.05, by the number of tests, and that gives you the corrected significance threshold.
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alpha
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Welch’s
the Welch
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Normalised tissue
unclear
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s 2
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different samples
reactions
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For the analysis,the technical replicates were assumed to be biological replicates
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ligation
the ligation
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from
by
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Direct
The direct
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in a
at
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thus
so that
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between methylated und unmethylated CpGs
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theirdiscrimination
to discriminate between methylated und unmethylated CpGs
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diverse conversion of cytosines
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more
orders of magnitude more
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Whereupon
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s e
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sodium-bisulfite
sodium metabisulfite
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in a
at
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Protocol
What you did not do due to lack of time: optimisation of the qRT-PCR assays. That would have included a melt curve analysis to detect primer dimers and/or off-target amplicons, and to then run the same reaction again, but with an additional "read" step during each cycle at a higher temperature than the 55C annealing temperature. The "read" temperature is chosen based on the melt curve so that primer dimers and smaller off-target amplicons have melted (become ssDNA) while the target still is dsDNA. This gives you more target-specific fluorescence measurements.
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amount
the amount
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Endogenous
The endogenous
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of
for
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defined
targeted
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threshold
the threshold
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There is only fluorescence if the dye bindsto the PCR product.
Any dsDNA will generate fluorescence, including, for example, primer dimers and off-target amplicons. That is why SYBR is considered relatively non-specific. But its is relatively cheap compared to more specific assays like TaqMan.
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The point where the fluorescence becomes measurable is calledthreshold cycleor quantification cycle.
Fluorescence always is measurable. The threshold cycle is when fluorescence exceeds a given and fixed threshold value, which is chosen to intersect the amplification curves during their exponential phase.
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after
during
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Input
The input
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SuperScript
The SuperScript
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e a
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appropriate
an appropriate
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e a
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d t
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The ratio 260nm/280nm was used to assess the purity of the DNA.
redundant
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m g
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RNase
the RNase
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RNeasy
the RNAeasy
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in consideration of
see above
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in consideration of
with consideration to
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Wavy bumps indicatethe activity (read depth, the number of reads).
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data
depth-of-sequencing-normalised and log-transformed read coverage data
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strand
strand of
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middle top
centre
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mentioned
shown
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statistical
the statistical
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. c
'e.g.' also is a prepositional phrase, i.e., needs to be followed by a comma
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. E
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. Z
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. C
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yt
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mentioned
shown
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t t
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On
In
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with
of
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RNA-seq data
scatter
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Genome
The genome
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UCSC
The UCSC
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terminate
extend
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across
extending across
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(activity is defined by the expression levels of the transcripts)
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activity
transcriptional activity
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Further requirements were
A further requirement was
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e a
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which means that onlythe intragenicpromoter influencestissue-specific gene transcription
reducing the potential of tissue-specific host promoter activity confounding the observations
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for
of
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to conduct
for
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RNA-seq data
scatter
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RNA-seq data
scatter
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s
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blue square and the red diamond are overlapping
put in parenthesis
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, which means that the
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m,
where the CGI is inactive and
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2c
where the CGI is highly active
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across
going across
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higher CGI promoter activity and
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(blue box)
(low blue data point)
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upstream
terminating upstream
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(red diamond)
(high red data point)
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terminating
going
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across
going across
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(lower left cornerof the figure)
(low x value)
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(red diamond in the bottom right cornerindicate transcripts across)
(large difference in y values: high blue versus low red data point)
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(upper right cornerof the figure)
(large x value)
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transcripts
amount of fragments spanning
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transcripts terminating
(scaled) amount of cDNA fragments mapping
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RNA-seq data
scatter
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from a large difference to a non-existing difference in absoluteterms
no difference between across and upstream
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terminating across than upstream
extending across than terminating upstream
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(red line)
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a negative correlation with the data
fewer transcripts extending
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pattern
scatter plot pattern
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in theRNA-seq dataplot
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out of 632
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single
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and forall 30 mouse tissues
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RNA-seq data plots were
A scatter plot of intragenic CGI activity versus the upstream and across quantities for all 30 tissues was
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provided
consisted of
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scalingfora comparable dataset
scaling across tissues to make quantities comparable
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distinguished
made non-redundant
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of
to
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Pearson correlation coefficient was calculated to compare the expression level of the intragenic promoter (CGI) or transcriptionalactivity initiating at the intragenic CGI and the ratio upstream/across, which means transcription upstream and splicing across the CGI. For the negative control sample dataset Pearson correlation coefficient was calculated as well with simulated intragenic promoters. The cut-off was set close to the maximum correlation of the negative control dataset and defined at 0.5.
redundant
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drift
bias
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and defined
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of
observed for
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For the negative control sample dataset,Pearson correlation coefficient was calculated alongside simulated intragenic promoters.
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compare
relate
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Pearson
The Pearson
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and splicing
versus transcription
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conducting a permutation test for resampling of the dataset
randomly permuting the intragenic CGI activities across tissues
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‘Host gene upstream of the intragenic promoter’(Upstream) defined as fragment numbers measured from the TSS until the beginning of the CGI and ‘Gene through the intragenic promoter’(Across), defined as exonic fragmentsoverlapping with the host gene;
redundant
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fragment
aligned sequenced cDNA fragments
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overlapping with the host gene
spanning across the intragenic CGI
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exonic
host gene exon-anchored
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sense and antisense orientation
separately for the sense and antisense orientations of transcription from the CGI relative to the host gene
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mouse
the mouse
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hybrid cross Mus m. musculus(B6) and Mus m. castaneus(Cast) -BxC or hybrid cross Mus m. castaneus(Cast)and Mus m. musculus(B6)–CxB
you may be asked if some of the inconsistencies between your results and the RNAseq data can be explained by the genetic differences between the hybrids and the B6 mice used by ENCODE. possibly yes, since Cast is quite distant from B6 in evolutionary terms, i.e., there are many genetic differences that definitely have an effect on gene expression. the next question may be: well, why then did you use hybrid tissue? it's the only option available for some tissues, given our tissue stocks, and we don't have a B6 colony any more.
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list
a list
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source
sources
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CGIs
CGI transcriptional states
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with those from
between
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maternal and paternal allele
the maternal and paternal alleles
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gene
locus
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lab
group (lab is slang)
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Work form
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has recently
not so recent
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mechanisms
marks
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Investigating the utilisation of alternative polyadenylation sites at the H13/Mcts23imprinted locus will shed light on this matter.
has been done and published by Andrew Wood and Michael Cowley
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However, an allele-specific epigenetic control for mechanisms such as polyadenylation has yet to be proven.
H13 and Herc3 are proof
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TSS
TSSs
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DNMTs interact with enzymes that regulategene expression andaretypicallyinvolved ingene repression27.
out of context
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for
is for
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nucleosomes
nucleosome = octamer of histones
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resulting in small fractions called
forming
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Notably, CGIs within gene bodies show the highest variationof DNA methylation between different tissues and somatic cells
redundant?
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of
with
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, notwithstanding of gene expression
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X
the X
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s
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CG
GC
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a
the
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Dnmt1 copies
during cell division
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paternal
template
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DNA methylation
in vertebrates (in bacteria, 5mA is common)
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RNA-binding sites
transcription factor binding sites
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over
relative to
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there may be different methylation patterns
too vague
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ym
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and occurs if an intron is retained,which normally would be removed
redundant
-
which
that instead of which
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post
co- rather than post-
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it
what is 'it'?
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ExceptforCGI2,where a simultaneousenrichment of H3K4me2 (chromatin mark associated with active transcription) and H3K27me3(silencing chromatin modification) has beendetectedin all somatic tissues, which resultsin bivalent chromatin. In brain,no enrichment of the chromatin marks wasfound.
ungrammatical and hence, unclear
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(1C, 1B1 and 1B2)
remove parenthesis
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s i
-
the
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e
-
e
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juston few
for only a few
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due
due to
-
-most exon
3'UTR
-
cause
can cause
-
s a
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variances
variants
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non-histonic
indeed, genes encoding histones are treated very differently
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constituting
consisting
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cell/tissue-specific combinations
cell/tissue-type
-
-specific
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Each gene generates
Most genes generate
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hence a specific set
specific sets
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As soon as the stop codon has been transcribed
stop codon is not responsible for transcription termination and polyadenylation. signals in the sequence of the transcribed RNA and the DNA are responsible, such as the polyadenylation site, which has an RNA consensus sequence of AAUAAA.
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gi
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do not cause
are not caused by
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heritable
mitotically or meiotically heritable
-
accepted
typically understood
-
p t
-
these considerations
the hypothesis
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On the hypothesis
Hypothesising
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murine
redundant
-
sa
missing comma following prepositional phrase
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na
-
am
auf dem?
-
e
-
am
auf dem?
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- Dec 2015
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www.google.com www.google.com
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web PDF
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