Progress on Chapter 2 results

thesis.lyx

@@ -1215,7 +1215,7 @@ ChIP-seq blacklisting is important
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@@ -1234,6 +1234,14 @@ status collapsed
 \begin_inset Caption Standard
 
 \begin_layout Plain Layout
+
+\series bold
+\begin_inset CommandInset label
+LatexCommand label
+name "fig:CCF-with-blacklist"
+
+\end_inset
+
 Cross-correlation plots with blacklisted reads removed
 \end_layout
 
@@ -1251,7 +1259,7 @@ Cross-correlation plots with blacklisted reads removed
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+status open
 
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@@ -1270,6 +1278,14 @@ status collapsed
 \begin_inset Caption Standard
 
 \begin_layout Plain Layout
+
+\series bold
+\begin_inset CommandInset label
+LatexCommand label
+name "fig:CCF-without-blacklist"
+
+\end_inset
+
 Cross-correlation plots without removing blacklisted reads
 \end_layout
 
@@ -1281,6 +1297,199 @@ Cross-correlation plots without removing blacklisted reads
 \end_inset
 
 
+\end_layout
+
+\begin_layout Subsection
+ChIP-seq peak calling
+\end_layout
+
+\begin_layout Standard
+\begin_inset Flex TODO Note (inline)
+status open
+
+\begin_layout Plain Layout
+Replace these figures with a single table of # of peaks called at chosen
+ IDR threshold, showing that SICER has more
+\end_layout
+
+\end_inset
+
+
+\end_layout
+
+\begin_layout Standard
+\begin_inset Float figure
+wide false
+sideways false
+status open
+
+\begin_layout Plain Layout
+\begin_inset Flex TODO Note (inline)
+status open
+
+\begin_layout Plain Layout
+Re-generate IDR rank consistency plots for SICER and MACS side-by-side
+\end_layout
+
+\end_inset
+
+
+\end_layout
+
+\begin_layout Plain Layout
+\begin_inset Caption Standard
+
+\begin_layout Plain Layout
+
+\series bold
+\begin_inset CommandInset label
+LatexCommand label
+name "fig:IDR-RC-H3K4me2"
+
+\end_inset
+
+Irreproducible Discovery Rate consistency plots for H3K4me2
+\end_layout
+
+\end_inset
+
+
+\end_layout
+
+\end_inset
+
+
+\end_layout
+
+\begin_layout Standard
+\begin_inset Float figure
+wide false
+sideways false
+status open
+
+\begin_layout Plain Layout
+\begin_inset Flex TODO Note (inline)
+status open
+
+\begin_layout Plain Layout
+Re-generate IDR rank consistency plots for SICER and MACS side-by-side
+\end_layout
+
+\end_inset
+
+
+\end_layout
+
+\begin_layout Plain Layout
+\begin_inset Caption Standard
+
+\begin_layout Plain Layout
+
+\series bold
+\begin_inset CommandInset label
+LatexCommand label
+name "fig:IDR-RC-H3K4me3"
+
+\end_inset
+
+Irreproducible Discovery Rate consistency plots for H3K4me3
+\end_layout
+
+\end_inset
+
+
+\end_layout
+
+\end_inset
+
+
+\end_layout
+
+\begin_layout Standard
+\begin_inset Float figure
+wide false
+sideways false
+status open
+
+\begin_layout Plain Layout
+\begin_inset Flex TODO Note (inline)
+status open
+
+\begin_layout Plain Layout
+Re-generate IDR rank consistency plots for SICER and MACS side-by-side
+\end_layout
+
+\end_inset
+
+
+\end_layout
+
+\begin_layout Plain Layout
+\begin_inset Caption Standard
+
+\begin_layout Plain Layout
+
+\series bold
+\begin_inset CommandInset label
+LatexCommand label
+name "fig:IDR-RC-H3K27me3"
+
+\end_inset
+
+Irreproducible Discovery Rate consistency plots for H3K27me3
+\end_layout
+
+\end_inset
+
+
+\end_layout
+
+\end_inset
+
+
+\end_layout
+
+\begin_layout Standard
+Figures 
+\begin_inset CommandInset ref
+LatexCommand ref
+reference "fig:IDR-RC-H3K4me2"
+plural "false"
+caps "false"
+noprefix "false"
+
+\end_inset
+
+, 
+\begin_inset CommandInset ref
+LatexCommand ref
+reference "fig:IDR-RC-H3K4me3"
+plural "false"
+caps "false"
+noprefix "false"
+
+\end_inset
+
+, and 
+\begin_inset CommandInset ref
+LatexCommand ref
+reference "fig:IDR-RC-H3K27me3"
+plural "false"
+caps "false"
+noprefix "false"
+
+\end_inset
+
+ show the IDR rank-consistency plots for peaks called in an arbitrarily-chosen
+ pair of donors.
+ For all 3 histone marks, when the peaks for each donor are ranked according
+ to their scores, SICER produces much more reproducible results between
+ donors.
+ This is consistent with SICER's stated goal of identifying broad peaks,
+ in contrast to MACS, which is designed for identifying sharp peaks.
+ Based on this observation, the SICER peak calls were used for all downstream
+ analyses that involved ChIP-seq peaks.
+ 
 \end_layout
 
 \begin_layout Subsection
@@ -1423,8 +1632,30 @@ ChIP-seq must be corrected for hidden confounding factors
 status open
 
 \begin_layout Plain Layout
-Consolidate these into 1 2x3 grid.
+Consolidate figures 
+\begin_inset CommandInset ref
+LatexCommand ref
+reference "fig:PCoA-H3K4me2-bad"
+plural "false"
+caps "false"
+noprefix "false"
+
+\end_inset
+
+ through 
+\begin_inset CommandInset ref
+LatexCommand ref
+reference "fig:PCoA-H3K27me3-good"
+plural "false"
+caps "false"
+noprefix "false"
+
+\end_inset
+
+ into 1 2x3 grid lettered A through F.
  For now, just refer to them as if they were a single figure.
+ Also, do the good PCoA plots belong in the results section since they are
+ also used there?
 \end_layout
 
 \end_inset
@@ -1480,7 +1711,7 @@ PCoA plot of H3K4me2 windows, before subtracting surrogate variables
 \begin_inset Float figure
 wide false
 sideways false
-status collapsed
+status open
 
 \begin_layout Plain Layout
 \align center
@@ -1568,7 +1799,7 @@ PCoA plot of H3K4me3 windows, before subtracting surrogate variables
 \begin_inset Float figure
 wide false
 sideways false
-status collapsed
+status open
 
 \begin_layout Plain Layout
 \align center
@@ -1656,7 +1887,7 @@ PCoA plot of H3K27me3 windows, before subtracting surrogate variables
 \begin_inset Float figure
 wide false
 sideways false
-status collapsed
+status open
 
 \begin_layout Plain Layout
 \align center
@@ -1697,7 +1928,7 @@ PCoA plot of H3K27me3 windows, after subtracting surrogate variables
 \end_layout
 
 \begin_layout Itemize
-Figures showing BCV plots with and without SVA for each histone mark.
+Figures showing BCV plots with and without SVA for each histone mark?
 \end_layout
 
 \begin_layout Standard
@@ -2104,168 +2335,22 @@ H3K4 and H3K27 methylation occur in broad regions and are enriched near
 \end_layout
 
 \begin_layout Standard
-\begin_inset Flex TODO Note (inline)
-status open
-
-\begin_layout Plain Layout
-Replace these figures with a single table of # of peaks called at chosen
- IDR threshold, showing that SICER has more
-\end_layout
-
-\end_inset
-
-
-\end_layout
-
-\begin_layout Standard
-\begin_inset Float figure
+\begin_inset Float table
 wide false
 sideways false
 status open
 
 \begin_layout Plain Layout
+\align center
 \begin_inset Flex TODO Note (inline)
 status open
 
 \begin_layout Plain Layout
-Re-generate IDR rank consistency plots for SICER and MACS side-by-side
-\end_layout
-
-\end_inset
-
-
-\end_layout
-
-\begin_layout Plain Layout
-\begin_inset Caption Standard
-
-\begin_layout Plain Layout
-
-\series bold
-\begin_inset CommandInset label
-LatexCommand label
-name "fig:IDR-RC-H3K4me2"
-
-\end_inset
-
-Irreproducible Discovery Rate consistency plots for H3K4me2
-\end_layout
-
-\end_inset
-
-
-\end_layout
-
-\end_inset
-
-
-\end_layout
-
-\begin_layout Standard
-\begin_inset Float figure
-wide false
-sideways false
-status open
-
-\begin_layout Plain Layout
-\begin_inset Flex TODO Note (inline)
-status open
-
-\begin_layout Plain Layout
-Re-generate IDR rank consistency plots for SICER and MACS side-by-side
-\end_layout
-
-\end_inset
-
-
-\end_layout
-
-\begin_layout Plain Layout
-\begin_inset Caption Standard
-
-\begin_layout Plain Layout
-
-\series bold
-\begin_inset CommandInset label
-LatexCommand label
-name "fig:IDR-RC-H3K4me3"
-
-\end_inset
-
-Irreproducible Discovery Rate consistency plots for H3K4me3
-\end_layout
-
-\end_inset
-
-
-\end_layout
-
-\end_inset
-
-
-\end_layout
-
-\begin_layout Standard
-\begin_inset Float figure
-wide false
-sideways false
-status open
-
-\begin_layout Plain Layout
-\begin_inset Flex TODO Note (inline)
-status open
-
-\begin_layout Plain Layout
-Re-generate IDR rank consistency plots for SICER and MACS side-by-side
-\end_layout
-
-\end_inset
-
-
-\end_layout
-
-\begin_layout Plain Layout
-\begin_inset Caption Standard
-
-\begin_layout Plain Layout
-
-\series bold
-\begin_inset CommandInset label
-LatexCommand label
-name "fig:IDR-RC-H3K27me3"
-
-\end_inset
-
-Irreproducible Discovery Rate consistency plots for H3K27me3
-\end_layout
-
-\end_inset
-
-
-\end_layout
-
-\end_inset
-
-
-\end_layout
-
-\begin_layout Standard
-\begin_inset Float table
-wide false
-sideways false
-status open
-
-\begin_layout Plain Layout
-\align center
-\begin_inset Flex TODO Note (inline)
-status open
-
-\begin_layout Plain Layout
-Need 
-\emph on
-median
-\emph default
- peak width, not mean
+Also get 
+\emph on
+median
+\emph default
+ peak width and maybe other quantiles (25%, 75%)
 \end_layout
 
 \end_inset
@@ -2324,7 +2409,7 @@ genome coverage
 \begin_inset Text
 
 \begin_layout Plain Layout
-read coverage
+FRiP
 \end_layout
 
 \end_inset
@@ -2490,7 +2575,12 @@ name "tab:peak-calling-summary"
 
 \end_inset
 
-SICER+IDR peak-calling summary
+Peak-calling summary.
+ 
+\series default
+For each histone mark, the number of peaks called using SICER at an IDR
+ threshold of ???, the mean width of those peaks, the fraction of the genome
+ covered by peaks, and the fraction of reads in peaks (FRiP).
 \end_layout
 
 \end_inset
@@ -2504,56 +2594,49 @@ SICER+IDR peak-calling summary
 \end_layout
 
 \begin_layout Standard
-Figures 
-\begin_inset CommandInset ref
-LatexCommand ref
-reference "fig:IDR-RC-H3K4me2"
-plural "false"
-caps "false"
-noprefix "false"
-
-\end_inset
-
-, 
+Table 
 \begin_inset CommandInset ref
 LatexCommand ref
-reference "fig:IDR-RC-H3K4me3"
+reference "tab:peak-calling-summary"
 plural "false"
 caps "false"
 noprefix "false"
 
 \end_inset
 
-, and 
+ gives a summary of the peak calling statistics for each histone mark.
+ Consistent with previous observations [CITATION NEEDED], all 3 histone
+ marks occur in broad regions spanning many consecutive nucleosomes, rather
+ than in sharp peaks as would be expected for a transcription factor or
+ other molecule that binds to specific sites.
+ This conclusion is further supported by Figure 
 \begin_inset CommandInset ref
 LatexCommand ref
-reference "fig:IDR-RC-H3K27me3"
+reference "fig:CCF-with-blacklist"
 plural "false"
 caps "false"
 noprefix "false"
 
 \end_inset
 
- show the IDR rank-consistency plots for peaks called in an arbitrarily-chosen
- pair of donors.
- For all 3 histone marks, when the peaks for each donor are ranked according
- to their scores, SICER produces much more reproducible results between
- donors.
- This is consistent with SICER's stated goal of identifying broad peaks,
- in contrast to MACS, which is designed for identifying sharp peaks.
- Based on this observation, the SICER peak calls were used for all downstream
- analyses that involved ChIP-seq peaks.
- Table 
+, in which a clear nucleosome-sized periodicity is visible in the cross-correlat
+ion value for each sample, indicating that each time a given mark is present
+ on one histone, it is also likely to be found on adjacent histones as well.
+ H3K27me3 enrichment in particular is substantially more broad than either
+ H3K4 mark, with a mean peak width of almost 19,000 bp.
+ This is also reflected in the periodicity observed in Figure 
 \begin_inset CommandInset ref
 LatexCommand ref
-reference "tab:peak-calling-summary"
+reference "fig:CCF-with-blacklist"
 plural "false"
 caps "false"
 noprefix "false"
 
 \end_inset
 
- gives a summary of the peak calling statistics for each histone mark.
+, which remains strong much farther out for H3K27me3 than the other marks,
+ showing H3K27me3 especially tends to be found on long runs of consecutive
+ histones.
 \end_layout
 
 \begin_layout Standard
@@ -2563,12 +2646,12 @@ sideways false
 status open
 
 \begin_layout Plain Layout
-\align center
-\begin_inset Graphics
-	filename graphics/CD4-csaw/Promoter Peak Distance Profile-PAGE1-CROP.pdf
-	lyxscale 50
-	width 100col%
-	groupId colwidth
+\begin_inset Flex TODO Note (inline)
+status open
+
+\begin_layout Plain Layout
+Ensure this figure uses the peak calls from the new analysis.
+\end_layout
 
 \end_inset
 
@@ -2576,18 +2659,31 @@ status open
 \end_layout
 
 \begin_layout Plain Layout
-\begin_inset Caption Standard
+\begin_inset Flex TODO Note (inline)
+status open
 
 \begin_layout Plain Layout
-
-\series bold
-\begin_inset CommandInset label
-LatexCommand label
-name "fig:effective-promoter-radius"
+Need a control: shuffle all peaks and repeat, N times.
+ Do real vs shuffled control both in a top/bottom arrangement.
+\end_layout
 
 \end_inset
 
-Enrichment of peaks in promoter neighborhoods.
+
+\end_layout
+
+\begin_layout Plain Layout
+\begin_inset Flex TODO Note (inline)
+status open
+
+\begin_layout Plain Layout
+Consider counting TSS inside peaks as negative number indicating how far
+ 
+\emph on
+inside
+\emph default
+ the peak the TSS is (i.e.
+ distance to nearest non-peak area).
 \end_layout
 
 \end_inset
@@ -2596,7 +2692,21 @@ Enrichment of peaks in promoter neighborhoods.
 \end_layout
 
 \begin_layout Plain Layout
+\begin_inset Flex TODO Note (inline)
+status open
+
+\begin_layout Plain Layout
+The H3K4 part of this figure is included in 
+\begin_inset CommandInset citation
+LatexCommand cite
+key "LaMere2016"
+literal "false"
+
+\end_inset
 
+ as Fig.
+ S2.
+ Do I need to do anything about that?
 \end_layout
 
 \end_inset
@@ -2604,94 +2714,928 @@ Enrichment of peaks in promoter neighborhoods.
 
 \end_layout
 
-\begin_layout Itemize
-Each histone mark is enriched within a certain radius of gene TSS positions,
- but that radius is different for each mark (figure 
-\begin_inset CommandInset ref
-LatexCommand ref
-reference "fig:effective-promoter-radius"
-plural "false"
-caps "false"
-noprefix "false"
+\begin_layout Plain Layout
+\align center
+\begin_inset Graphics
+	filename graphics/CD4-csaw/Promoter Peak Distance Profile-PAGE1-CROP.pdf
+	lyxscale 50
+	width 80col%
 
 \end_inset
 
-, previously in 
-\begin_inset CommandInset citation
-LatexCommand cite
-key "LaMere2016"
-literal "false"
+
+\end_layout
+
+\begin_layout Plain Layout
+\begin_inset Caption Standard
+
+\begin_layout Plain Layout
+
+\series bold
+\begin_inset CommandInset label
+LatexCommand label
+name "fig:near-promoter-peak-enrich"
 
 \end_inset
 
- Fig.
- S2)
+Enrichment of peaks in promoter neighborhoods.
+ 
+\series default
+This plot shows the distribution of distances from each annotated transcription
+ start site in the genome to the nearest called peak.
+ Each line represents one combination of histone mark, cell type, and time
+ point.
+ Distributions are smoothed using kernel density estimation [CITE?].
+ Transcription start sites that occur 
+\emph on
+within
+\emph default
+ peaks were excluded from this plot to avoid a large spike at zero that
+ would overshadow the rest of the distribution.
 \end_layout
 
-\begin_layout Subsection
-H3K4 and H3K27 promoter methylation has broadly the expected correlation
- with gene expression
+\end_inset
+
+
+\end_layout
+
+\end_inset
+
+
 \end_layout
 
 \begin_layout Standard
-\begin_inset Flex TODO Note (inline)
+\begin_inset Float table
+wide false
+sideways false
 status open
 
 \begin_layout Plain Layout
-This section can easily be cut, especially if I can't find those plots.
+\align center
+\begin_inset Tabular
+<lyxtabular version="3" rows="4" columns="2">
+<features tabularvalignment="middle">
+<column alignment="center" valignment="top">
+<column alignment="center" valignment="top">
+<row>
+<cell alignment="center" valignment="top" topline="true" bottomline="true" leftline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+Histone mark
+\end_layout
+
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" bottomline="true" leftline="true" rightline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+Effective promoter radius
+\end_layout
+
+\end_inset
+</cell>
+</row>
+<row>
+<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+H3K4me2
+\end_layout
+
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" leftline="true" rightline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+1 kb
+\end_layout
+
+\end_inset
+</cell>
+</row>
+<row>
+<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+H3K4me3
+\end_layout
+
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" leftline="true" rightline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+1 kb
+\end_layout
+
+\end_inset
+</cell>
+</row>
+<row>
+<cell alignment="center" valignment="top" topline="true" bottomline="true" leftline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+H3K27me3
+\end_layout
+
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" bottomline="true" leftline="true" rightline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+2.5 kb
+\end_layout
+
+\end_inset
+</cell>
+</row>
+</lyxtabular>
+
+\end_inset
+
+
+\end_layout
+
+\begin_layout Plain Layout
+\begin_inset Caption Standard
+
+\begin_layout Plain Layout
+
+\series bold
+\begin_inset CommandInset label
+LatexCommand label
+name "tab:effective-promoter-radius"
+
+\end_inset
+
+Effective promoter radius for each histone mark.
+
+\series default
+ These values represent the approximate distance from transcription start
+ site positions within which an excess of peaks are found, as shown in Figure
+ 
+\begin_inset CommandInset ref
+LatexCommand ref
+reference "fig:near-promoter-peak-enrich"
+plural "false"
+caps "false"
+noprefix "false"
+
+\end_inset
+
+.
+\end_layout
+
+\end_inset
+
+
+\end_layout
+
+\begin_layout Plain Layout
+
+\end_layout
+
+\end_inset
+
+
+\end_layout
+
+\begin_layout Standard
+\begin_inset Flex TODO Note (inline)
+status open
+
+\begin_layout Plain Layout
+Problem: the effective promoter radius concept is an interesting result
+ on its own, hence its placement here.
+ However, it is also important in the methods section, which comes first.
+ What do? Refer forward to this section? Move this section to Methods?
+\end_layout
+
+\end_inset
+
+
+\end_layout
+
+\begin_layout Standard
+All 3 histone marks tend to occur more often near promoter regions, as shown
+ in Figure 
+\begin_inset CommandInset ref
+LatexCommand ref
+reference "fig:near-promoter-peak-enrich"
+plural "false"
+caps "false"
+noprefix "false"
+
+\end_inset
+
+.
+ The majority of each density distribution is flat, representing the background
+ density of peaks genome-wide.
+ Each distribution has a peak near zero, representing an enrichment of peaks
+ close transcription start site (TSS) positions relative to the remainder
+ of the genome.
+ Interestingly, the 
+\begin_inset Quotes eld
+\end_inset
+
+radius
+\begin_inset Quotes erd
+\end_inset
+
+ within which this enrichment occurs is not the same for every histone mark
+ (Table 
+\begin_inset CommandInset ref
+LatexCommand ref
+reference "tab:effective-promoter-radius"
+plural "false"
+caps "false"
+noprefix "false"
+
+\end_inset
+
+).
+ For H3K4me2 and H3K4me3, peaks are most enriched within 1
+\begin_inset space ~
+\end_inset
+
+kbp of TSS positions, while for H3K27me3, enrichment is broader, extending
+ to 2.5
+\begin_inset space ~
+\end_inset
+
+kbp.
+ These 
+\begin_inset Quotes eld
+\end_inset
+
+effective promoter radii
+\begin_inset Quotes erd
+\end_inset
+
+ were used for all further promoter-based analyses.
+\end_layout
+
+\begin_layout Standard
+\begin_inset Flex TODO Note (inline)
+status open
+
+\begin_layout Plain Layout
+Consider also showing figure for distance to nearest peak center, and reference
+ median peak size once that is known.
+\end_layout
+
+\end_inset
+
+
+\end_layout
+
+\begin_layout Subsection
+H3K4 and H3K27 promoter methylation has broadly the expected correlation
+ with gene expression
+\end_layout
+
+\begin_layout Standard
+\begin_inset Flex TODO Note (inline)
+status open
+
+\begin_layout Plain Layout
+This section can easily be cut, especially if I can't find those plots.
+\end_layout
+
+\end_inset
+
+
+\end_layout
+
+\begin_layout Itemize
+H3K4 is correlated with higher expression, and H3K27 is correlated with
+ lower expression genome-wide
+\end_layout
+
+\begin_layout Standard
+\begin_inset Flex TODO Note (inline)
+status open
+
+\begin_layout Plain Layout
+Grr, gotta find these figures.
+ Maybe in the old analysis? At least one of these plots is definitely in
+ Sarah's paper.
+\end_layout
+
+\end_inset
+
+
+\end_layout
+
+\begin_layout Itemize
+Figures showing these correlations: box/violin plots of expression distributions
+ with every combination of peak presence/absence in promoter
+\end_layout
+
+\begin_layout Itemize
+Appropriate statistical tests showing significant differences in expected
+ directions
+\end_layout
+
+\begin_layout Subsection
+RNA-seq and H3K4 methylation patterns in naive and memory show convergence
+ at day 14
+\end_layout
+
+\begin_layout Standard
+\begin_inset Float figure
+wide false
+sideways false
+status collapsed
+
+\begin_layout Plain Layout
+\align center
+\begin_inset Graphics
+	filename graphics/CD4-csaw/ChIP-seq/H3K4me2-promoter-PCA-group-CROP.png
+	lyxscale 25
+	width 100col%
+	groupId colwidth-raster
+
+\end_inset
+
+
+\end_layout
+
+\begin_layout Plain Layout
+\begin_inset Caption Standard
+
+\begin_layout Plain Layout
+
+\series bold
+\begin_inset CommandInset label
+LatexCommand label
+name "fig:PCoA-H3K4me2-prom"
+
+\end_inset
+
+PCoA plot of H3K4me2 promoters, after subtracting surrogate variables
+\end_layout
+
+\end_inset
+
+
+\end_layout
+
+\end_inset
+
+
+\end_layout
+
+\begin_layout Standard
+\begin_inset Float figure
+wide false
+sideways false
+status collapsed
+
+\begin_layout Plain Layout
+\align center
+\begin_inset Graphics
+	filename graphics/CD4-csaw/ChIP-seq/H3K4me3-promoter-PCA-group-CROP.png
+	lyxscale 25
+	width 100col%
+	groupId colwidth-raster
+
+\end_inset
+
+
+\end_layout
+
+\begin_layout Plain Layout
+\begin_inset Caption Standard
+
+\begin_layout Plain Layout
+
+\series bold
+\begin_inset CommandInset label
+LatexCommand label
+name "fig:PCoA-H3K4me3-prom"
+
+\end_inset
+
+PCoA plot of H3K4me3 promoters, after subtracting surrogate variables
+\end_layout
+
+\end_inset
+
+
+\end_layout
+
+\end_inset
+
+
+\end_layout
+
+\begin_layout Standard
+\begin_inset Float figure
+wide false
+sideways false
+status collapsed
+
+\begin_layout Plain Layout
+\align center
+\begin_inset Graphics
+	filename graphics/CD4-csaw/ChIP-seq/H3K27me3-promoter-PCA-group-CROP.png
+	lyxscale 25
+	width 100col%
+	groupId colwidth-raster
+
+\end_inset
+
+
+\end_layout
+
+\begin_layout Plain Layout
+\begin_inset Caption Standard
+
+\begin_layout Plain Layout
+
+\series bold
+\begin_inset CommandInset label
+LatexCommand label
+name "fig:PCoA-H3K27me3-prom"
+
+\end_inset
+
+PCoA plot of H3K27me3 promoters, after subtracting surrogate variables
+\end_layout
+
+\end_inset
+
+
+\end_layout
+
+\end_inset
+
+
+\end_layout
+
+\begin_layout Standard
+\begin_inset Float figure
+wide false
+sideways false
+status collapsed
+
+\begin_layout Plain Layout
+\align center
+\begin_inset Graphics
+	filename graphics/CD4-csaw/RNA-seq/PCA-final-23-CROP.png
+	lyxscale 25
+	width 100col%
+	groupId colwidth-raster
+
+\end_inset
+
+
+\end_layout
+
+\begin_layout Plain Layout
+\begin_inset Caption Standard
+
+\begin_layout Plain Layout
+
+\series bold
+\begin_inset CommandInset label
+LatexCommand label
+name "fig:RNA-PCA-group"
+
+\end_inset
+
+RNA-seq PCoA showing principal coordiantes 2 and 3.
+\end_layout
+
+\end_inset
+
+
+\end_layout
+
+\end_inset
+
+
+\end_layout
+
+\begin_layout Standard
+\begin_inset Float table
+wide false
+sideways true
+status collapsed
+
+\begin_layout Plain Layout
+\align center
+\begin_inset Tabular
+<lyxtabular version="3" rows="6" columns="7">
+<features tabularvalignment="middle">
+<column alignment="center" valignment="top">
+<column alignment="center" valignment="top">
+<column alignment="center" valignment="top">
+<column alignment="center" valignment="top">
+<column alignment="center" valignment="top">
+<column alignment="center" valignment="top">
+<column alignment="center" valignment="top">
+<row>
+<cell alignment="center" valignment="top" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+
+\end_layout
+
+\end_inset
+</cell>
+<cell multicolumn="1" alignment="center" valignment="top" topline="true" leftline="true" rightline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+Number of significant promoters
+\end_layout
+
+\end_inset
+</cell>
+<cell multicolumn="2" alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+
+\end_layout
+
+\end_inset
+</cell>
+<cell multicolumn="2" alignment="center" valignment="top" topline="true" leftline="true" rightline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+
+\end_layout
+
+\end_inset
+</cell>
+<cell multicolumn="1" alignment="center" valignment="top" topline="true" leftline="true" rightline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+Est.
+ differentially modified promoters
+\end_layout
+
+\end_inset
+</cell>
+<cell multicolumn="2" alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+
+\end_layout
+
+\end_inset
+</cell>
+<cell multicolumn="2" alignment="center" valignment="top" topline="true" leftline="true" rightline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+
+\end_layout
+
+\end_inset
+</cell>
+</row>
+<row>
+<cell alignment="center" valignment="top" topline="true" bottomline="true" leftline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+Time Point
+\end_layout
+
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" bottomline="true" leftline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+H3K4me2
+\end_layout
+
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" bottomline="true" leftline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+H3K4me3
+\end_layout
+
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" bottomline="true" leftline="true" rightline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+H3K27me3
+\end_layout
+
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" bottomline="true" leftline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+H3K4me2
+\end_layout
+
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" bottomline="true" leftline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+H3K4me3
+\end_layout
+
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" bottomline="true" leftline="true" rightline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+H3K27me3
+\end_layout
+
+\end_inset
+</cell>
+</row>
+<row>
+<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+Day 0
+\end_layout
+
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+4553
+\end_layout
+
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+927
+\end_layout
+
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" leftline="true" rightline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+6
+\end_layout
+
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+9967
+\end_layout
+
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+4149
+\end_layout
+
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" leftline="true" rightline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+2404
+\end_layout
+
+\end_inset
+</cell>
+</row>
+<row>
+<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+Day 1
+\end_layout
+
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+567
+\end_layout
+
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+278
+\end_layout
+
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" leftline="true" rightline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+1570
+\end_layout
+
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+4370
+\end_layout
+
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+2145
+\end_layout
+
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" leftline="true" rightline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+6598
+\end_layout
+
+\end_inset
+</cell>
+</row>
+<row>
+<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+Day 5
+\end_layout
+
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+2313
+\end_layout
+
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+139
+\end_layout
+
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" leftline="true" rightline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+490
+\end_layout
+
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+9450
+\end_layout
+
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" leftline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+1148
+\end_layout
+
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" leftline="true" rightline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+4141
 \end_layout
 
 \end_inset
+</cell>
+</row>
+<row>
+<cell alignment="center" valignment="top" topline="true" bottomline="true" leftline="true" usebox="none">
+\begin_inset Text
 
-
+\begin_layout Plain Layout
+Day 14
 \end_layout
 
-\begin_layout Itemize
-H3K4 is correlated with higher expression, and H3K27 is correlated with
- lower expression genome-wide
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" bottomline="true" leftline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+0
 \end_layout
 
-\begin_layout Standard
-\begin_inset Flex TODO Note (inline)
-status open
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" bottomline="true" leftline="true" usebox="none">
+\begin_inset Text
 
 \begin_layout Plain Layout
-Grr, gotta find these figures.
- Maybe in the old analysis?
+0
 \end_layout
 
 \end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" bottomline="true" leftline="true" rightline="true" usebox="none">
+\begin_inset Text
 
-
+\begin_layout Plain Layout
+0
 \end_layout
 
-\begin_layout Itemize
-Figures showing these correlations: box/violin plots of expression distributions
- with every combination of peak presence/absence in promoter
-\end_layout
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" bottomline="true" leftline="true" usebox="none">
+\begin_inset Text
 
-\begin_layout Itemize
-Appropriate statistical tests showing significant differences in expected
- directions
+\begin_layout Plain Layout
+0
 \end_layout
 
-\begin_layout Subsection
-Naive-to-memory convergence observed in H3K4 and RNA-seq data, not in H3K27me3
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" bottomline="true" leftline="true" usebox="none">
+\begin_inset Text
+
+\begin_layout Plain Layout
+0
 \end_layout
 
-\begin_layout Standard
-\begin_inset Float figure
-wide false
-sideways false
-status open
+\end_inset
+</cell>
+<cell alignment="center" valignment="top" topline="true" bottomline="true" leftline="true" rightline="true" usebox="none">
+\begin_inset Text
 
 \begin_layout Plain Layout
-\align center
-\begin_inset Graphics
-	filename graphics/CD4-csaw/RNA-seq/PCA-final-23-CROP.png
-	lyxscale 25
-	width 100col%
-	groupId colwidth-raster
+0
+\end_layout
+
+\end_inset
+</cell>
+</row>
+</lyxtabular>
 
 \end_inset
 
@@ -2706,11 +3650,25 @@ status open
 \series bold
 \begin_inset CommandInset label
 LatexCommand label
-name "fig:RNA-PCA-group"
+name "tab:Number-signif-promoters"
 
 \end_inset
 
-RNA-seq PCoA showing principal coordiantes 2 and 3.
+Number of differentially modified promoters between naive and memory cells
+ at each time point after activation.
+ 
+\series default
+This table shows both the number of differentially modified promoters detected
+ at a 10% FDR threshold (left half), and the total number of differentially
+ modified promoters as estimated using the method of 
+\begin_inset CommandInset citation
+LatexCommand cite
+key "Phipson2013"
+literal "false"
+
+\end_inset
+
+ (right half).
 \end_layout
 
 \end_inset
@@ -2723,12 +3681,11 @@ RNA-seq PCoA showing principal coordiantes 2 and 3.
 
 \end_layout
 
-\begin_layout Itemize
-H3K4 and RNA-seq data show clear evidence of naive convergence with memory
- between days 1 and 5 (Figures 
+\begin_layout Standard
+Figures 
 \begin_inset CommandInset ref
 LatexCommand ref
-reference "fig:PCoA-H3K4me2-good"
+reference "fig:PCoA-H3K4me2-prom"
 plural "false"
 caps "false"
 noprefix "false"
@@ -2738,7 +3695,7 @@ noprefix "false"
 , 
 \begin_inset CommandInset ref
 LatexCommand ref
-reference "fig:PCoA-H3K4me3-good"
+reference "fig:PCoA-H3K4me3-prom"
 plural "false"
 caps "false"
 noprefix "false"
@@ -2748,7 +3705,24 @@ noprefix "false"
 , and 
 \begin_inset CommandInset ref
 LatexCommand ref
-reference "fig:RNA-PCA-group"
+reference "fig:PCoA-H3K27me3-prom"
+plural "false"
+caps "false"
+noprefix "false"
+
+\end_inset
+
+ show the patterns of variation in all 3 histone marks in the promoter regions
+ of the genome using principal coordinate analysis.
+ All 3 marks show a noticeable convergence between the naive and memory
+ samples at day 14, visible as an overlapping of the day 14 groups on each
+ plot.
+ This is consistent with the counts of significantly differentially modified
+ promoters and estimates of the total numbers of differentially modified
+ promoters shown in Table 
+\begin_inset CommandInset ref
+LatexCommand ref
+reference "tab:Number-signif-promoters"
 plural "false"
 caps "false"
 noprefix "false"
@@ -2756,37 +3730,113 @@ noprefix "false"
 \end_inset
 
 .
-\end_layout
+ For all histone marks, evidence of differential modification between naive
+ and memory samples was detected at every time point except day 14.
+ The day 14 convergence pattern is also present in the RNA-seq data (Figure
+ 
+\begin_inset CommandInset ref
+LatexCommand ref
+reference "fig:RNA-PCA-group"
+plural "false"
+caps "false"
+noprefix "false"
 
-\begin_layout Itemize
-Table of numbers of genes different between N & M at each time point, showing
- dwindling differences at later time points, consistent with convergence
-\end_layout
+\end_inset
 
-\begin_layout Itemize
-Similar figure for H3K27me3 showing lack of convergence (Figure 
+), albiet in the 2nd and 3rd principal coordinates, indicating that it is
+ not the most dominant pattern driving gene expression.
+ Taken together, the data show that promoter histone methylation for these
+ 3 histone marks and RNA expression for naive and memory cells are most
+ similar at day 14, the furthest time point after activation.
+ MOFA was also able to capture this day 14 convergence pattern in latent
+ factor 5 (Figure 
 \begin_inset CommandInset ref
 LatexCommand ref
-reference "fig:PCoA-H3K27me3-good"
+reference "fig:mofa-lf-scatter"
 plural "false"
 caps "false"
 noprefix "false"
 
 \end_inset
 
-)
+), which accounts for shared variation across all 3 histone marks and the
+ RNA-seq data, confirming that this is a coordinated pattern across all
+ 4 data sets.
+\end_layout
+
+\begin_layout Standard
+\begin_inset Flex TODO Note (inline)
+status collapsed
+
+\begin_layout Plain Layout
+This result feels shallow, somehow.
+ Am I oversimplifying the observation, or trivializing the amount of work
+ it took to get here? Shouldn't this section be more than one paragraph?
+ Am I just forgetting some supporting evidence that should also go here
+ in order to build up to the result? Or is it good that I have a simple
+ relatively straightforward result that doesn't take to long to explain,
+ and I'm just overthinking it?
+\end_layout
+
+\end_inset
+
+
+\end_layout
+
+\begin_layout Standard
+\begin_inset ERT
+status collapsed
+
+\begin_layout Plain Layout
+
+
+\backslash
+FloatBarrier
+\end_layout
+
+\end_inset
+
+
 \end_layout
 
 \begin_layout Subsection
 Effect of promoter coverage upstream vs downstream of TSS
 \end_layout
 
+\begin_layout Standard
+\begin_inset Flex TODO Note (inline)
+status open
+
+\begin_layout Plain Layout
+For the figures in this section, the group labels are arbitrary, so if time
+ allows, it would be good to manually reorder them in a logical way, e.g.
+ most upstream to most downstream.
+\end_layout
+
+\end_inset
+
+
+\end_layout
+
 \begin_layout Standard
 \begin_inset Float figure
 wide false
 sideways false
 status open
 
+\begin_layout Plain Layout
+\begin_inset Flex TODO Note (inline)
+status open
+
+\begin_layout Plain Layout
+These really need to be sub-figures A, B, and C.
+\end_layout
+
+\end_inset
+
+
+\end_layout
+
 \begin_layout Plain Layout
 \align center
 \begin_inset Graphics
@@ -2812,7 +3862,46 @@ name "fig:H3K4me2-neighborhood-clusters"
 
 \end_inset
 
-RNA-seq PCoA showing principal coordiantes 2 and 3.
+K-means clustering of promoter H3K4me2 relative coverage depth in naive
+ day 0 samples.
+ 
+\series default
+H3K4me2 ChIP-seq reads were binned into 500-bp windows tiled across each
+ promoter from 5
+\begin_inset space ~
+\end_inset
+
+kbp upstream to 5
+\begin_inset space ~
+\end_inset
+
+kbp downstream, and the logCPM values were normalized within each promoter
+ to an average of 0, yielding relative coverage depths.
+ These were then grouped using K-means clustering with 
+\begin_inset Formula $K=6$
+\end_inset
+
+, and the average bin values were plotted for each cluster.
+ The 
+\begin_inset Formula $x$
+\end_inset
+
+-axis is the genomic coordinate of each bin relative to the the transcription
+ start site, and the 
+\begin_inset Formula $y$
+\end_inset
+
+-axis is the mean relative coverage depth of that bin across all promoters
+ in the cluster.
+ Each line represents the average 
+\begin_inset Quotes eld
+\end_inset
+
+shape
+\begin_inset Quotes erd
+\end_inset
+
+ of the promoter coverage for promoters in that cluster.
 \end_layout
 
 \end_inset
@@ -2856,7 +3945,23 @@ name "fig:H3K4me2-neighborhood-pca"
 
 \end_inset
 
-RNA-seq PCoA showing principal coordiantes 2 and 3.
+PCA of promoter H3K4me2 relative coverage depth in naive day 0 samples,
+ colored by K-means cluster membership.
+ 
+\series default
+PCA was performed on the same relative promoter coverage values used for
+ K-means clustering in Figure 
+\begin_inset CommandInset ref
+LatexCommand ref
+reference "fig:H3K4me2-neighborhood-clusters"
+plural "false"
+caps "false"
+noprefix "false"
+
+\end_inset
+
+, and the first two principal components were plotted, coloring each point
+ by its K-means cluster identity.
 \end_layout
 
 \end_inset
@@ -2900,7 +4005,21 @@ name "fig:H3K4me2-neighborhood-expression"
 
 \end_inset
 
-RNA-seq PCoA showing principal coordiantes 2 and 3.
+Gene expression distributions grouped by H3K4me2 promoter coverage K-means
+ clustering.
+ 
+\series default
+For each of the clusters in Figure 
+\begin_inset CommandInset ref
+LatexCommand ref
+reference "fig:H3K4me2-neighborhood-clusters"
+plural "false"
+caps "false"
+noprefix "false"
+
+\end_inset
+
+
 \end_layout
 
 \end_inset
@@ -3099,6 +4218,16 @@ Try to boil it down to 3 main messages to get across
 \end_layout
 
 \begin_deeper
+\begin_layout Itemize
+Naive-to-memory convergence in certain data sets but not others, implies
+ which marks are involved in memory differentiation
+\end_layout
+
+\end_deeper
+\begin_layout Subsection
+Effective promoter radius 
+\end_layout
+
 \begin_layout Itemize
 "Promoter radius" is not constant and must be defined empirically for a
  given data set.
@@ -3106,15 +4235,15 @@ Try to boil it down to 3 main messages to get across
 \end_layout
 
 \begin_layout Itemize
-Naive-to-memory convergence in certain data sets but not others, implies
- which marks are involved in memory differentiation
+Further study required to demonstarte functional consequences of effective
+ promoter radius (e.g.
+ show diminished association with gene expression outside radius)
 \end_layout
 
-\begin_layout Itemize
-TSS positional coverage, hints of something interesting but no clear conclusions
+\begin_layout Subsection
+Convergence
 \end_layout
 
-\end_deeper
 \begin_layout Standard
 \begin_inset Float figure
 wide false
@@ -3178,8 +4307,16 @@ H3K27me3, canonically regarded as a deactivating mark, seems to have a more
 \end_layout
 
 \end_deeper
+\begin_layout Subsection
+Positional
+\end_layout
+
 \begin_layout Itemize
-TSS positional coverage 
+TSS positional coverage, hints of something interesting but no clear conclusions
+\end_layout
+
+\begin_layout Subsection
+Workflow
 \end_layout
 
 \begin_layout Standard
@@ -3236,6 +4373,10 @@ Decision-making based on trying every option and running the workflow downstream
 \end_layout
 
 \end_deeper
+\begin_layout Subsection
+Data quality issues limit conclusions
+\end_layout
+
 \begin_layout Chapter
 Improving array-based analyses of transplant rejection by optimizing data
  preprocessing
@@ -6577,7 +7718,7 @@ noprefix "false"
 \begin_inset Float table
 wide false
 sideways false
-status collapsed
+status open
 
 \begin_layout Plain Layout
 \align center
@@ -6811,7 +7952,15 @@ noprefix "false"
 \end_inset
 
 , the table shows the number of probes estimated to be differentially methylated
- between TX and the other 3 transplant statuses.
+ between TX and the other 3 transplant statuses using the method of 
+\begin_inset CommandInset citation
+LatexCommand cite
+key "Phipson2013"
+literal "false"
+
+\end_inset
+
+.
 \end_layout
 
 \end_inset
@@ -9715,6 +10864,20 @@ Check bib entry formatting & sort order
 \end_inset
 
 
+\end_layout
+
+\begin_layout Standard
+\begin_inset Flex TODO Note (inline)
+status open
+
+\begin_layout Plain Layout
+Check in-text citation format.
+ Probably don't just want [1], [2], etc.
+\end_layout
+
+\end_inset
+
+
 \end_layout
 
 \begin_layout Standard