Epigenetic regulation of brain region-specific microglia clearance activity

In the paper

“Only uniquely mapped reads were retained, and the alignments were subsequently filtered using the SAMtools package (v0.1.19)88 to remove duplicate reads.”

What the sources say

• Supported

  Section 2.2, “SAMtools software package,” identifies SAMtools as software for manipulating alignments and explicitly says it can remove PCR duplicates. The citation is attached to SAMtools in the citing sentence; the choice to retain only uniquely mapped reads describes the citing authors’ workflow, not a claim attributed to this source. (LLM, conf=0.91)

  cites [88] Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009). source

In the paper

“We used the negative binomial (NB) model implemented in edgeR (3.10.0)79–83 for our single cell differential expression analysis to explicitly account for the sparse count matrix”

What the sources say

• Partly supported

  The Introduction and Section 2.3 describe a negative binomial model for count-based differential-expression analysis of SAGE tags. Section 7.1 also considers zero counts, but the paper does not establish that an edgeR model accounts for a sparse single-cell count matrix; its setting is SAGE libraries, not single-cell data. (LLM, conf=0.86)

  cites [79] Robinson, M. D. & Smyth, G. K. Small-sample estimation of negative binomial dispersion, with applications to SAGE data. Biostatistics 9, 321–332 (2008). sourceExperimental — accuracy not yet measured
• Partly supported

  Section 2.2 describes a negative binomial model for differential expression of tag counts, and §4.3 discusses handling tags with zero counts in SAGE data. The paper does not establish that edgeR 3.10.0 uses this model to explicitly account for a sparse single-cell count matrix; its application is to replicated tag-count libraries, not single-cell data. (LLM, conf=0.88)

  cites [80] Robinson, M. D. & Smyth, G. K. Moderated statistical tests for assessing differences in tag abundance. Bioinformatics 23, 2881–2887 (2007). sourceExperimental — accuracy not yet measured
• Partly supported

  The Model section states that edgeR models count data with a negative binomial distribution for differential expression analysis. It does not address single-cell data or say that the model explicitly accounts for a sparse count matrix; it describes dispersion as accounting for biological variation between samples. (LLM, conf=0.87)

  cites [81] Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010). sourceExperimental — accuracy not yet measured
• Partly supported

  The Discussion and Results (‘Linear models for multifactor experiments’; page not provided) describe negative-binomial models for RNA-seq read counts implemented in edgeR. They do not establish that the model explicitly accounts for sparse single-cell count matrices; the paper discusses biological and technical variation in RNA-seq experiments instead. (LLM, conf=0.87)

  cites [82] McCarthy, D. J., Chen, Y. & Smyth, G. K. Differential expression analysis of multifactor RNA-seq experiments with respect to biological variation. Nucleic Acids Res. 40, 4288–4297 (2012). sourceExperimental — accuracy not yet measured
• Partly supported

  The Introduction identifies edgeR as an implementation of a negative binomial model for count-based differential expression, and Materials and Methods describes the NB model used for read counts. Our copy does not establish that this model explicitly accounts for sparsity in a single-cell count matrix; the Introduction describes it as accounting for biological variability in genome-scale count data. Page numbers are unavailable in our copy. (LLM, conf=0.87)

  cites [83] Zhou, X., Lindsay, H. & Robinson, M. D. Robustly detecting differential expression in RNA sequencing data using observation weights. Nucleic Acids Res. 42, e91 (2014). sourceExperimental — accuracy not yet measured

In the paper

“leading to impaired motor responses, to decreased learning and memory, and to the development of anxiety and seizures in mice.”

In the paper

“Furthermore, cbMg show an increase in the expression of genes characteristic of immature microglia9,10, as well as genes that are expressed in neurodegenerative disease-associated microglia (such as primed microglia, MGnD, and DAMs)11–13 (Supplementary Fig. 5c,d, Supplementary Table 4).”

What the sources say

• Not checked yetcites [9] Mass, E. et al. Specification of tissue-resident macrophages during organogenesis. Science 353, aaf4238 (2016). source
• Not checked yetcites [10] Matcovitch-Natan, O. et al. Microglia development follows a stepwise program to regulate brain homeostasis. Science 353, aad8670 (2016). source
• Not checked yetcites [11] Mathys, H. et al. Temporal tracking of microglia activation in neurodegeneration at single-cell resolution. Cell Rep. 21, 366–380 (2017). source
• Not checked yetcites [12] Wang, Y. et al. TREM2 lipid sensing sustains the microglial response in an Alzheimer’s disease model. Cell 160, 1061–1071 (2015). source
• Not checked yetcites [13] Krasemann, S. et al. The TREM2-APOE pathway drives the transcriptional phenotype of dysfunctional microglia in neurodegenerative diseases. Immunity 47, 566–581.e9 (2017). source

In the paper

“cbMg in the adult mouse brain show a primed/phagocytic microglia phenotype11–13 characterized by a less ramified morphology, reduced branching sites (Supplementary Fig. 1a), decreased cell volume (Supplementary Fig. 1b,c), and an increase in CD68+ lysosome content (Fig. 1a, Supplementary Fig. 1b,c).”

What the sources say

• Not checked yetcites [11] Mathys, H. et al. Temporal tracking of microglia activation in neurodegeneration at single-cell resolution. Cell Rep. 21, 366–380 (2017). source
• Not checked yetcites [12] Wang, Y. et al. TREM2 lipid sensing sustains the microglial response in an Alzheimer’s disease model. Cell 160, 1061–1071 (2015). source
• Not checked yetcites [13] Krasemann, S. et al. The TREM2-APOE pathway drives the transcriptional phenotype of dysfunctional microglia in neurodegenerative diseases. Immunity 47, 566–581.e9 (2017). source

In the paper

“A large fraction of the cbMg-enriched genes, including Apoe, Axl, Colec12, Cd74, Lilrb4, Mrc1, and Ms4a7 (Fig. 1d,e, Supplementary Fig. 5a,b, Supplementary Table 3), encode proteins involved in apoptotic cell detection, engulfment, clearance of cell debris, and lipid/protein catabolism and have been shown to support clearance activity in phagocytic macrophages in peripheral tissues21.”

What the sources say

• Not checked yetcites [21] Blander, J. M. The many ways tissue phagocytes respond to dying cells. Immunol. Rev. 277, 158–173 (2017). source

In the paper

“Expression of the clearance-associated proteins AXL, ApoE, CD74, MHCII, and MRC1 is significantly higher in cbMg, while actin polymerization-regulating FSCN1 protein expression shows a significant enrichment in stMg (Fig. 2c, Supplementary Fig. 7d).”

In the paper

“The changes in PRC2-deficient cbMg gene expression patterns are associated with enhanced cbMg-ramification, a reduction in cbMg lysosomal content (Fig. 6f), and an increased number of cCASP3+ cells in the cerebellum (Fig. 6g).”

In the paper

“Notably, the anxiety and seizure phenotypes are not observed in one-year-old wild-type mice that live in the chronic absence of microglia (continuous PLX treatment starting 1 month of age, Supplementary Fig. 13h-j)”

In the paper

“Most of these down-regulated genes are H3K27me3-negative and are, therefore, not directly controlled by PRC2 in stMg (Fig. 5e).”

In the paper

“PRC2 deficiency in cbMg, on the other hand, has no significant impact on cerebellar Purkinje cell spine numbers (Supplementary Fig. 12e) and cerebellum-dependent behaviors (Supplementary Fig. 12f).”

In the paper

“The shift of stMg from a surveillance state toward a more clearance-promoting cbMg-like state (Fig. 6a,b), while not affecting overall microglia numbers or morphology (Fig. 6c, Supplementary Fig. 11i), is associated with a significant increase in the stMg lysosomal content (Fig. 6c).”

In the paper

“Like in the striatum, we found that loss of PRC2 activity in cortical microglia (cxMg) is associated with a similar switch towards a microglia clearance phenotype (Supplementary Fig. 12g), including an increase in cxMg lysosomal content (Supplementary Fig. 12h).”

In the paper

“Deficiency in Eed has no effect on the overall size and morphology of the striatum (data not shown), MSN density (Supplementary Fig. 12a), or survival as defined by the presence of cCASP3+ cells (Supplementary Fig. 12b).”

In the paper

“Co-labeling with different cell type-specific markers revealed that the majority of the apoptotic cells are of neuronal origin and are distributed within all layers of the cerebellum (Supplementary Fig. 9a,b).”

In the paper

“We found that deficiency in Eed and the ensuing loss of H3K27me3 had no immediate impact on microglia activation but leads to rather moderate and progressive changes in stMg gene expression between 3-9 months of age (Fig. 5e,f).”

In the paper

“Likewise, during an elevated plus maze paradigm, mutant mice spend significantly more time in the closed compartment and significantly less time in the more stressful open compartment as compared to their littermate controls (Fig. 7d, Supplementary Fig. 13b,c).”

In the paper

“While young Cx3cr1CreErt2/+;Eedfl/fl and control mice did not display any notable seizure events prior to 10 months of age, we detected an increased frequency of handling-induced tonic-clonic seizure episodes in aged mutant mice (Fig. 7e, Supplementary Fig. 13f).”

In the paper

“This exposure to dying cells was associated with the time-dependent induction of cbMg-enriched transcription factors (Fig. 3c, Supplementary Fig. 10c) as well as the down-regulation of stMg enriched homeostatic surveillance genes (Fig. 3c, Supplementary Fig. 10d,e) in the absence of pro-inflammatory gene activation (Supplementary Fig. 10f).”

In the paper

“The deficiency in Eed and the ensuing loss of H3K27me3 led to the up-regulation of several known PRC2 targets, such as Tbx15 and Hoxd830,31 (Fig. 5g,h, Supplementary Fig. 11f,g, Supplementary Table 8,9), but did not affect the expression of genes that define microglia lineage identity (Supplementary Fig. 11f,g, Supplementary Table 8,9) or genes characteristic of microglia pro-inflammatory”

What the sources say

• Not checked yetcites [30] Jadhav, U. et al. Acquired tissue-specific promoter bivalency is a basis for PRC2 necessity in adult cells. Cell 165, 1389–1400 (2016). source
• Not checked yetcites [31] von Schimmelmann, M. et al. Polycomb repressive complex 2 (PRC2) silences genes responsible for neurodegeneration. Nat. Neurosci. 19, 1321–1330 (2016). source

In the paper

“Accordingly, loss of PRC2 in cbMg, while following a similar temporal pattern of progressive PRC2 target gene induction (Fig. 5f, Supplementary Fig. 11e), is associated with the up-regulation of selected stMg-enriched genes and the down-regulation of cbMg-enriched genes (Fig. 6d,e).”

In the paper

“These transcription factors are also induced upon exposure to apoptotic cells as well as in the Eed-deficient stMg, which acquire an aberrant clearance phenotype (Supplementary Fig. 14b).”

In the paper

“Accordingly, we found that both the deletion of microglia as well as the attenuation of their cell clearance capability are associated with the accumulation of dying cells in the cerebellum but not in the striatum or the cortex.”

In the paper

“Moreover, exposure of forebrain microglia to dying cells in vitro increases the expression of the H3K27me3-specific demethylases, Kdm6a/b, and is associated with the progressive activation of cbMg-enriched clearance genes.”

In the paper

“All cells were positive for Cx3cr1, Csf1r, Hexb, or Tmem119.”

In the paper

“Staurosporine-treated Jurkat cells were ~70% positive for phosphatidylserine staining and ~95% negative for propidium iodide staining identifying the major population as early apoptotic (Supplementary Fig. 7e).”

In the paper

“SW p-value was < 0.0001 for all samples.”

In the paper

“Here we show that microglia clearance activity in the adult brain is regionally regulated and depends on the rate of neuronal attrition.”

In the paper

“The cerebellar microglia phenotype is reminiscent of microglia during early brain development9,10 as well as microglia associated with neurodegenerative disease11–13.”

What the sources say

• Not checked yetcites [9] Mass, E. et al. Specification of tissue-resident macrophages during organogenesis. Science 353, aaf4238 (2016). source
• Not checked yetcites [10] Matcovitch-Natan, O. et al. Microglia development follows a stepwise program to regulate brain homeostasis. Science 353, aad8670 (2016). source
• Not checked yetcites [11] Mathys, H. et al. Temporal tracking of microglia activation in neurodegeneration at single-cell resolution. Cell Rep. 21, 366–380 (2017). source
• Not checked yetcites [12] Wang, Y. et al. TREM2 lipid sensing sustains the microglial response in an Alzheimer’s disease model. Cell 160, 1061–1071 (2015). source
• Not checked yetcites [13] Krasemann, S. et al. The TREM2-APOE pathway drives the transcriptional phenotype of dysfunctional microglia in neurodegenerative diseases. Immunity 47, 566–581.e9 (2017). source

In the paper

“Development and ageing of the brain are associated with the attrition and loss of neurons1.”

What the sources say

• Not checked yetcites [1] Dickstein, D. L., Weaver, C. M., Luebke, J. I. & Hof, P. R. Dendritic spine changes associated with normal aging. Neuroscience 251, 21–32 (2013). source

In the paper

“One of the brain regions displaying the most pronounced age-dependent neuronal loss is the cerebellum, where neuronal numbers start declining at the end of adolescence3,4.”

What the sources say

• Not checked yetcites [3] Woodruff-Pak, D. S. et al. Differential effects and rates of normal aging in cerebellum and hippocampus. Proc. Natl Acad. Sci. USA 107, 1624–1629 (2010). source
• Not checked yetcites [4] Morterá, P. & Herculano-Houzel, S. Age-related neuronal loss in the rat brain starts at the end of adolescence. Front. Neuroanat. 6, 45 (2012). source

In the paper

“Importantly, we show that the TRAP approach not only allows for the region-specific analysis of microglia-enriched mRNA expression (Supplementary Fig. 3, Supplementary Table 1) but also precludes nonspecific microglia activation and concurrent up-regulation of immediate early and pro-inflammatory genes that occur during commonly used microglia isolation approaches (Supplementary Fig. 4, Supplement”

In the paper

“In addition to its known role in autophagy22, LC3 has been recently identified as a novel regulator of rapid apoptotic cell clearance in macrophages in a process termed LC3-associated phagocytosis (LAP), which occurs in the absence of immune activation23.”

What the sources say

• Not checked yetcites [22] Galluzzi, L. et al. Molecular definitions of autophagy and related processes. EMBO J. 36, 1811–1836 (2017). source
• Not checked yetcites [23] Heckmann, B. L., Boada-Romero, E., Cunha, L. D., Magne, J. & Green, D. R. LC3-associated phagocytosis and inflammation. J. Mol. Biol. 429, 3561–3576 (2017). source

In the paper

“While the age-dependent loss of cerebellar mass and neuronal numbers3,4 implies ongoing cell death, we could not detect dying cells in the cerebellum of adult wild-type mice.”

What the sources say

• Not checked yetcites [3] Woodruff-Pak, D. S. et al. Differential effects and rates of normal aging in cerebellum and hippocampus. Proc. Natl Acad. Sci. USA 107, 1624–1629 (2010). source
• Not checked yetcites [4] Morterá, P. & Herculano-Houzel, S. Age-related neuronal loss in the rat brain starts at the end of adolescence. Front. Neuroanat. 6, 45 (2012). source

In the paper

“which indicates that the behavioral changes are unlikely due to a general loss of microglia function but rather reflect the aberrant microglia clearance activity in the striatum and cortex.”

In the paper

“Microglia-specific Eed ablation was induced by tamoxifen treatment between 4-6 weeks of age in order to exclude any impact of PRC2 inactivation on microglia development (Fig. 5a).”

In the paper

“KDM6A/B mediate the removal of the suppressive chromatin modification H3K27me3, which is associated with silenced genes26.”

What the sources say

• Not checked yetcites [26] Swigut, T. & Wysocka, J. H3K27 demethylases, at long last. Cell 131, 29–32 (2007). source

In the paper

“The induction of behavioral locomotor sensitization in response to cocaine depends on the structural plasticity of MSNs and is associated with lasting changes in MSN spine density32.”

What the sources say

• Not checked yetcites [32] Nestler, E. J. The neurobiology of cocaine addiction. Sci. Pract. Perspect. 3, 4–10 (2005). source

In the paper

“Disorders accompanied by alterations in spine numbers, including neurodegenerative and neuropsychiatric disorders, are frequently associated with the development of anxiety and seizures33 and cognitive decline34.”

What the sources say

• Not checked yetcites [33] Friedman, D., Honig, L. S. & Scarmeas, N. Seizures and epilepsy in Alzheimer’s disease. CNS Neurosci. Ther. 18, 285–294 (2012). source
• Not checked yetcites [34] Hong, S. et al. Complement and microglia mediate early synapse loss in Alzheimer mouse models. Science 352, 712–716 (2016). source

In the paper

“H3K27me3 is mediated by PRC2, which has a well-established role in the regulation of cell differentiation during development16.”

What the sources say

• Not checked yetcites [16] Margueron, R. & Reinberg, D. The polycomb complex PRC2 and its mark in life. Nature 469, 343–349 (2011). source

In the paper

“Enrichment of mRNAs encoding the H3K27me3-specific demethylases, KDM6A/B26 in cbMg (Supplementary Fig. 5b, Supplementary Table 3) as well as the up-regulation of Kdm6a/b in microglia upon exposure to dying cells in vitro (Fig. 3c, Supplementary Fig. 10g) point to the possible role of PRC2/H3K27me3 in suppressing clearance-promoting genes in stMg.”

What the sources say

• Not checked yetcites [26] Swigut, T. & Wysocka, J. H3K27 demethylases, at long last. Cell 131, 29–32 (2007). source

In the paper

“Collectively, our data point to a largely selective role of PRC2 in the maintenance of regional microglia specification in the adult brain, where it controls the cell-intrinsic suppression of an aberrant pro-phagocytic/clearance-like phenotype in stMg in vivo.”

In the paper

“The possible causal role of H3K27me3 in suppressing clearance-promoting genes in microglia is supported at several levels.”

In the paper

“Recent evidence suggests that microglia can sense and clear apoptotic cells at the earliest stages of apoptosis25.”

What the sources say

• Not checked yetcites [25] Fourgeaud, L. et al. TAM receptors regulate multiple features of microglial physiology. Nature 532, 240–244 (2016). source

In the paper

“On the other hand, increased activation of the MEK-ERK pathway has been linked to Ezh2 overexpression48.”

What the sources say

• Not checked yetcites [48] Fujii, S. et al. RAS oncogenic signal upregulates EZH2 in pancreatic cancer. Biochem. Biophys. Res. Commun. 417, 1074–1079 (2012). source

In the paper

“This process, often referred to as efferocytosis, is a multi-step process: (i) the recognition of “eat-me” signals presented by apoptotic cells, (ii) engulfment of cells/debris, (iii) degradation of engulfed material, and (iv) excretion of metabolic products21.”

What the sources say

• Not checked yetcites [21] Blander, J. M. The many ways tissue phagocytes respond to dying cells. Immunol. Rev. 277, 158–173 (2017). source

In the paper

“While the loss of PRC2 activity is associated with reduced MEK-ERK activation in ES cells47, the inhibition of MEK-ERK activity leads to attenuated PRC2 chromatin association on PRC2 target genes in these cells47.”

What the sources say

• Not checked yetcites [47] Tee, W.-W., Shen, S. S., Oksuz, O., Narendra, V. & Reinberg, D. Erk1/2 activity promotes chromatin features and RNAPII phosphorylation at developmental promoters in mouse ESCs. Cell 156, 678–690 (2014). source

In the paper

“Our data also show that microglia clearance function, while important for the removal of dying cells, must be tightly controlled in other brain regions to avoid microglia-mediated damage of healthy neurons.”

In the paper

“The lack of inflammatory gene activation in cbMg suggests that the microglia clearance activity may antagonize/suppress the activation of inflammatory genes in a fashion that has been observed in peripheral macrophages21,23.”

What the sources say

• Not checked yetcites [21] Blander, J. M. The many ways tissue phagocytes respond to dying cells. Immunol. Rev. 277, 158–173 (2017). source
• Not checked yetcites [23] Heckmann, B. L., Boada-Romero, E., Cunha, L. D., Magne, J. & Green, D. R. LC3-associated phagocytosis and inflammation. J. Mol. Biol. 429, 3561–3576 (2017). source

In the paper

“The ability of aberrant intrinsically activated microglia to cause neuronal damage is further supported by recent findings showing that cell-autonomous activation of the RAF–MEK–ERK pathway in microglia leads to the loss of synapses and the development of a severe late-onset neurodegenerative disorder7.”

What the sources say

• Not checked yetcites [7] Mass, E. et al. A somatic mutation in erythro-myeloid progenitors causes neurodegenerative disease. Nature 549, 389–393 (2017). source

In the paper

“While most of the newly generated neurons/T-cells undergo apoptotic cell death, the dying cells cannot be readily detected due to their rapid removal by resident macrophages/microglia25,39.”

What the sources say

• Not checked yetcites [25] Fourgeaud, L. et al. TAM receptors regulate multiple features of microglial physiology. Nature 532, 240–244 (2016). source
• Not checked yetcites [39] Dzhagalov, I. L., Chen, K. G., Herzmark, P. & Robey, E. A. Elimination of self-reactive T cells in the thymus: a timeline for negative selection. PLoS Biol. 11, e1001566 (2013). source

In the paper

“recent studies using a small molecule-based inhibitor specifically targeting KDM6A/B demethylase activity revealed that H3K27 demethylation, and more specifically KDM6A/B catalytic activity, are critical determinants of pro-inflammatory gene activation in human primary macrophages28.”

What the sources say

• Not checked yetcites [28] Kruidenier, L. et al. A selective jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response. Nature 488, 404–408 (2012). source

In the paper

“Based on these data, it is plausible that clearance gene expression in microglia is controlled in a KDM6A/B/H3K27me3-dependent fashion and that the induction of clearance genes is associated with a targeted reduction in H3K27me3 levels at specific clearance gene loci in vivo.”

In the paper

“All subjects correspond to data points within two standard deviations from the sample mean, and no subjects were excluded from the behavioral analyses.”

In the paper

“The ChIP-seq data was first checked for quality using the various metrics generated by FastQC (v0.11.2; http://www.bioinformatics.babraham.ac.uk/projects/fastqc).”

In the paper

“No statistical methods were used to pre-determine sample sizes but our sample sizes are similar to those reported in previous publications19,31,95,99,108.”

In the paper

“For all behavioral experiments, a mixed population of males and females with sex- and age-matched corresponding littermate control animals were used.”

In the paper

“Tg(Vav1-iCre), Tg(Csf1r-iCre), and Tg(Csf1r-Cre/Esr1*) lines were excluded due to neuronal targeting, while the Cx3cr1CreErt2/+(Jung) line was excluded due to insufficient targeting of microglia.”

In the paper

“and the outliers were further justified by the aforementioned quality control metrics as well as experimental metadata.”

In the paper

“No randomization protocol was used.”

In the paper

“In brief, exon 6 was chosen for gene deletion as a single base pair mutation in this exon was previously shown to disrupt the interaction of EED with EZH1/2 proteins, thus resulting in an Eed-null phenotype61,62.”

What the sources say

• Not checked yetcites [61] Schumacher, A., Faust, C. & Magnuson, T. Positional cloning of a global regulator of anterior-posterior patterning in mice. Nature 384, 648 (1996). source
• Not checked yetcites [62] Denisenko, O., Shnyreva, M., Suzuki, H. & Bomsztyk, K. Point mutations in the WD40 domain of Eed block its interaction with Ezh2. Mol. Cell. Biol. 18, 5634–5642 (1998). source

In the paper

“The quality of the library pools was assessed by 2200 TapeStation (Agilent).”

In the paper

“Normal distribution was assessed by the Shapiro-Wilk (SW) normality test.”

In the paper

“We included plates as covariates to account for confounding effect of individuals in the generalized linear model”

In the paper

“only samples with RIN>9 were considered for subsequent analysis.”

In the paper

“All mice used for experiments were backcrossed to the C57Bl/6 background for ≥ 5 generations.”

In the paper

“The data collection was randomized for all experiments.”

In the paper

“No statistical methods were used to pre-determine sample sizes but our sample sizes are similar to those reported in previous publications19,31,95,99,108.”

What the sources say

• Not checked yetcites [19] Heiman, M. et al. A translational profiling approach for the molecular characterization of CNS cell types. Cell 135, 738–748 (2008). source
• Not checked yetcites [31] von Schimmelmann, M. et al. Polycomb repressive complex 2 (PRC2) silences genes responsible for neurodegeneration. Nat. Neurosci. 19, 1321–1330 (2016). source
• Not checked yetcites [95] Schaefer, A. et al. Control of cognition and adaptive behavior by the GLP/G9a epigenetic suppressor complex. Neuron 64, 678–691 (2009). source

In the paper

“and the quasi-likelihood framework within edgeR to handle the pronounced variability of the single cell expression states.”

What the sources say

• Not checked yetcites [79] Robinson, M. D. & Smyth, G. K. Small-sample estimation of negative binomial dispersion, with applications to SAGE data. Biostatistics 9, 321–332 (2008). source
• Not checked yetcites [80] Robinson, M. D. & Smyth, G. K. Moderated statistical tests for assessing differences in tag abundance. Bioinformatics 23, 2881–2887 (2007). source
• Not checked yetcites [81] Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010). source
• Not checked yetcites [82] McCarthy, D. J., Chen, Y. & Smyth, G. K. Differential expression analysis of multifactor RNA-seq experiments with respect to biological variation. Nucleic Acids Res. 40, 4288–4297 (2012). source
• Not checked yetcites [83] Zhou, X., Lindsay, H. & Robinson, M. D. Robustly detecting differential expression in RNA sequencing data using observation weights. Nucleic Acids Res. 42, e91 (2014). source

In the paper

“This line was used as an alternative TRAP line to ensure that region-specific gene expression changes are not caused by Cx3cr1 haplo-insufficiency in the Cx3cr1CreErt2/+(Litt) mice.”

In the paper

“Cultures were regularly tested negative for mycoplasma using Lonza kit according to manufacturer’s instructions (MycoAlert LT-07).”

In the paper

“Cells with fewer than 100,000 mapped reads were removed.”

In the paper

“Experimenters were blinded during imaging and behavioral experiments.”

In the paper

“The Cx3cr1CreErt2/+(Litt)52 line was selected for all subsequent experiments due to its highly efficient (>99% of microglia) and microglia-specific Cre expression pattern throughout the brain.”

What the sources say

• Not checked yetcites [52] Parkhurst, C. N. et al. Microglia promote learning-dependent synapse formation through brain-derived neurotrophic factor. Cell 155, 1596–1609 (2013). source

In the paper

“Cells pretreated with 2 μM Cytochalasin D (Sigma-Aldrich, C8273) for 30 min before and during the incubation of microglia with pHrodo-EAJ were used as a negative control.”

In the paper

“Gender ratios and ages were matched within control and treatment groups.”

In the paper

“For antibody staining controls, primary antibody incubation was omitted, and instead slides were incubated with Alexa Fluor-conjugated secondary antibodies”

In the paper

“For TRAP from Eed-deficient microglia, Cx3cr1CreErt2/+(Litt);Eef1a1LSL.eGFPL10a/+;Eedfl/fl mice were compared to Cx3cr1CreErt2/+(Litt);Eef1a1LSL.eGFPL10a/+;Eedfl/+ sex- and age-matched littermate controls.”

In the paper

“Furthermore, aberrantly activated EZH2 was recently found to synergistically cooperate with hyperactive BRAF in the regulation of tumorigenesis49.”

What the sources say

• Not checked yetcites [49] Souroullas, G. P. et al. An oncogenic Ezh2 mutation induces tumors through global redistribution of histone 3 lysine 27 trimethylation. Nat. Med. 22, 632–640 (2016). source

In the paper

“Animals were allocated to treatment groups to ensure uniform distribution of ages and sexes in each group.”

In the paper

“Additionally, hierarchical clustering was used to assess the outliers once again to protect against false positives/negatives from PCA”

In the paper

“The expression of IRES-YFP did not interfere with microglia TRAP efficiency.”

In the paper

“Principal component analysis (PCA) was performed on the top 500 most variable genes across all samples based on the VST data to visually assess if there were any outliers.”

In the paper

“Cx3cr1+ perivascular/meningeal macrophages were identified by low Tmem119, low Hexb, and high Mrc1 expression35,84 and excluded from single cell analysis (n=1-5 per plate).”

What the sources say

• Not checked yetcites [35] Goldmann, T. et al. Origin, fate and dynamics of macrophages at central nervous system interfaces. Nat. Immunol. 17, 797–805 (2016). source
• Not checked yetcites [84] Bennett, M. L. et al. New tools for studying microglia in the mouse and human CNS. Proc. Natl. Acad. Sci. USA 113, E1738–E1746 (2016). source

In the paper

“Gene expression and protein analyses were not performed blind to the conditions of the experiments.”

In the paper

“Comparisons for Cx3cr1CreErt2/+(Litt);Eef1a1LSL.eGFPL10a/+;Eedfl/fl mice were carried out with age- and sex-matched control Cx3cr1CreErt2/+(Litt);Eef1a1LSL.eGFPL10a/+;Eedfl/+ mice.”

In the paper

“Increased EZH2 expression/activity is associated with a genome-wide redistribution of H3K27me3 that includes the loss of H3K27me3 and the transcriptional activation of numerous PRC2 suppressed genes in these cells49.”

What the sources say

• Not checked yetcites [49] Souroullas, G. P. et al. An oncogenic Ezh2 mutation induces tumors through global redistribution of histone 3 lysine 27 trimethylation. Nat. Med. 22, 632–640 (2016). source

In the paper

“C57Bl/6J wild-type mice were used to test for nonspecific mRNA enrichment.”

In the paper

“The read alignment, read counting as well as quality assessment using metrics such as total mapping rate, mitochondrial and ribosomal mapping rates were done in parallel using an in-house workflow pipeline called SPEctRA70.”

What the sources say

• Not checked yetcites [70] Purushothaman, I. & Shen, L. SPEctRA: First stable release. https://zenodo.org/record/60547#.WyweGlVKipp (2016).

In the paper

“A cohort of Cx3cr1CreErt2/+ and their respective Cx3cr1+/+ wild-type littermate controls were tested on selected behavior paradigms to exclude potential behavioral alterations due to Cx3cr1 haploinsufficiency in Cx3cr1CreErt2/+; Eedfl/fl mice.”