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Muroy et al. Neuroimmunol Neuroinflammation 2020;7:166-82 I http://dx.doi.org/10.20517/2347-8659.2020.16 Page 169

RNA extraction
Mice were sacrificed according to the approved protocol. Brains were quickly isolated and frontal cortical
areas were dissected, flash frozen, and stored at -80 °C. RNA was extracted using a bead homogenizer (30 s,
setting “5”; Bead Mill, VWR) in Trizol reagent (ThermoFisher, Waltham, MA). Total RNA was extracted using
the Direct-zol RNA miniprep kit (Zymo Research, Irvine, CA) according to the manufacturer’s instructions.
For cell lines, after immune stimulation, media was aspirated and wells were washed 2 × with ice-cold 1 ×
PBS (Invitrogen, Carlsbad, CA). RNA was extracted using the Direct-zol RNA miniprep kit (Zymo Research,
Irvine, CA).

RT-qPCR
TM
cDNA was reversed transcribed from total RNA using the SuperScript III First-Strand Synthesis System
kit (ThermoFisher, Waltham, MA) following the manufacturer’s instructions. RT-qPCR was run using
SYBR green (Roche, Pleasanton, CA) on a QuantStudio 6 (ThermoFisher, Waltham, MA) real-time PCR
machine. All RT-qPCR primers were specific to the desired template, spanned exon-exon junctions,
and captured all transcript variants for the specific gene under study. Ct values were normalized to the
housekeeping gene hypoxanthine phosphoribosyltransferase (Hprt). Primer sequences used in this study
are listed in Supplementary Table 1.

RNA-seq library preparation and analysis
RNA was extracted from a total of n = 3 replicates per condition (Phf15 KO or control) and was used to
prepare libraries for RNA sequencing using the mRNA HyperPrep Kit according to the manufacturer’s
instructions (KAPA Biosystems, Wilmington, MA). Libraries were quality control checked via Qubit
(ThermoFisher, Waltham, MA) and via RT-qPCR with a next generation sequencing library quantification
kit (Zymo Research, Irvine, CA). RNA sequencing (one lane) was performed on a HiSeq4000 sequencing
system (Illumina Inc., San Diego, CA; UC Berkeley Genomics Sequencing Laboratory). Sequencing reads
were aligned to the Mus musculus reference genome assembly GRCm38 (mm10) using Spliced Transcripts
[29]
Alignment to a Reference (STAR) aligner . Count data were analyzed with Hypergeometric Optimization
of Motif EnRichment (HOMER) software for next-generation sequencing analysis (http://homer.ucsd.edu/
[30]
homer/ngs/index.html), which uses the R/Bioconductor package DESeq2 to perform differential gene
expression analysis. To adjust for multiple comparisons, DESeq2 uses the Benjamini-Hochberg procedure
to control the false discovery rate and returned false discovery rate adjusted P values and log -fold
2
expression changes between Phf15 KO and control conditions for each gene. Genes were filtered by adjusted
P value (adjusted P < 0.01 for upregulated genes or 0.05 for downregulated genes) and log -fold change in
2
expression (greater than 1.5 log -fold change for upregulated genes and less than -1.5 for downregulated
2
genes). Too few downregulated genes (< 200) passed the more stringent adjusted P < 0.01 cutoff for robust
downstream biological function analysis, thus the adjusted P value threshold was lowered to P adj < 0.05.
[31]
Results were visualized using the R package EnhancedVolcano . Lists of upregulated and downregulated
[32]
genes were input into Metascape , a gene annotation and analysis tool, to determine enriched biological
themes within the gene lists.

Motif enrichment
Transcription factor binding site (“motif”) enrichment was analyzed using HOMER (http://homer.ucsd.
edu/homer/ngs/index.html).

Statistical analysis
Relative mRNA expression of Phf15 in mouse frontal cortical areas was analyzed using ordinary one-way
ANOVA with post hoc Tukey’s multiple comparisons to compare expression levels across age. Percent KD
and time course experiments measuring expression levels of inflammatory markers [Tnfα, nitric oxide
synthase, inducible (Nos2), and IL-1 β] between control and Phf15 shRNAs shPhf15-1 and shPhf15-2 after