GE Healthcare), and the recombinant proteins
were eluted, desalted, and concentrated.
In vitro DPR protein and HP1aassays
HP1aliquid droplets (90mM monomer) were
formed in buffer containing 50 mM tris, pH 7.5.
The preformed HP1adroplets were spotted onto
a coverslip and imaged for 5 min, and then
245 mM(PR) 8 or (PA) 8 peptides were added to the
sample and the sample was imaged for another
5 to 10 min.
i^3 N iPSC culture and neuronal
differentiation
We modified a well-characterized control iPSC
line (WTC11) that harbors a dox-inducible NGN2
transgene at the AAVS1 locus (i^3 N) ( 62 , 63 ).
dCas9-BFP-KRAB was stably expressed in these
i^3 N iPSCs via TALEN-mediated integration of a
CAG-dCas9-BFP-KRAB expression cassette into
theCLYBLsafeharborlocus( 55 ). The dCas9-
BFP-KRAB iPSCs were transduced with lentivi-
rus expressing HP1a-sgRNA for 3 days and then
selected by the addition of puromycin. To dif-
ferentiate i^3 N dCas9-BFP-KRAB iPSCs express-
ing HP1asgRNA into neurons, iPSCs were
dissociated by using Accutase (#AT-104, Inno-
vative Cell Technologies) and then seeded onto
dishes coated with Matrigel (354230, Corning).
Three days after differentiation, cells were dis-
sociated by using Accutase and then seeded
onto poly-L-ornithine–coated plates (6-well plate)
or glass coverslips (24-well plate) at a density
of 7 × 10^5 or 2 × 10^4 cells per well, respectively.
Sixdayslater,theneuronswerefixedwith4%
paraformaldehyde for immunofluorescence stain-
ing or harvested for Western blot and qPCR
analyses.
Statistics
Data are presented as the mean ± the standard
error of the mean (SEM) and analyzed with a
two-tailed unpairedttest or one-way or two-way
analysis of variance (ANOVA) followed by Tukey’s
post hoc analysis (Prism statistical software). End
points of interest [i.e., body weight, brain weight,
poly(PR)-positive cells, poly(PR) expression, NeuN-
positive cortical neurons, Purkinje cell density,
transgene RNA levels,GfapandCD68mRNA
expression, and Gfap and CD68 immunopositiv-
ity] were compared between male and female
mice within each cohort. Other than body weight,
no sex differences were observed. With the ex-
ception of body weight, analyses were performed
on all mice within a given cohort. Data are pres-
ented such as to distinguish male and female
mice;malemicearerepresentedbysolidsym-
bols on dot plots, and female mice by empty
symbols. Statistical analysis of RNA-seq data is
described in the section RNA-seq, Gene Ontol-
ogy, and RE analyses.
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