Antibodies
|
Rabbit monoclonal anti-CUG-BP1 |
Abcam |
RRID:AB_11141441; Cat #: ab129115 |
Rabbit polyclonal anti-hnRNP-K |
Bethyl |
RRID:AB_530281; Cat #: A300-678A |
Rabbit polyclonal anti-MATR3 |
Bethyl |
RRID:AB_2141651; Cat #: IHC-00081 |
Rabbit polyclonal anti-RYBP (DEDAF) |
Millipore Sigma |
RRID:AB_2285466; Cat #: AB3637 |
Rabbit monoclonal anti-EZH2 |
Cell signaling Technology |
RRID:AB_10694683; Cat #: 5246 |
Rabbit polyclonal anti-CIZ1 |
Novus Biologicals |
RRID:AB_1048573; Cat #: NB100-74624 |
Rabbit polyclonal anti-histone H3 phospho-Serine 10 |
Active Motif |
RRID:AB_2793206; Cat #: 39253 |
Rabbit polyclonal anti-GFP |
Abcam |
Cat #: ab6556 |
Donkey anti-rabbit IgG Alexa Fluor 488 |
Invitrogen |
RRID: AB_2535792; Cat #: A-21206 |
Donkey anti-rabbit IgG CF568 |
Sigma |
Cat #: SAB4600076 |
Goat anti-rabbit IgG Alexa Fluor 647 |
Life Technologies |
RRID: AB_2535813; Cat #: A21245 |
Bacterial and virus strains
|
Stellar Competent Cells |
Clontech |
Cat #: 636766 |
One Shot TOP10 Chemically Competent |
Thermo Fisher Scientific |
Cat #: C404003 |
10-beta Competent E.coli (High Efficiency) |
NEB |
Cat #: C3019H |
Biological samples
|
|
|
N/A |
|
|
Chemicals, peptides, and recombinant proteins
|
HaloTag Ligands for Super Resolution Microscopy JF 549 |
Promega |
Cat #: GA1111 |
HaloTag Ligands for Super Resolution Microscopy JF 646 |
Promega |
Cat #: GA1121 |
HaloTag TMR Ligand |
Promega |
Cat #: PRG8252 |
HaloLink Resin |
Promega |
Cat #: G1912 |
Aminoallyl dUTP |
Sigma-Aldrich |
Cat #: A 0410 |
ATTO 488-NHS Ester |
Sigma-Aldrich |
Cat #: 41698-1MG-F |
Alexa Fluor 568 NHS Ester |
Thermo Fisher Scientific |
Cat #: A20003 |
Cy3 Mono-NHS Ester |
VWR |
Cat #:PA13101 |
Cy5 NHS-Ester |
VWR |
Cat #: 95017-506 |
CF Dye Azide 568 |
Biotium |
Cat #: 92082 |
Texas Red-X, Succinimidyl Ester, mixed isomers |
Thermo Fisher Scientific |
Cat #: T6134 |
ProLong Live Antifade Reagent |
Thermo Fisher Scientific |
Cat #: P36975
|
DNase I recombinant, RNase-free |
Sigma-Aldrich |
Cat #: 4716728001 |
DNA Polymerase I (10 U/μL) |
Thermo Fisher Scientific |
Cat #: EP0042 |
Phusion High-Fidelity DNA Polymerase (2 U/μL) |
Thermo Fisher Scientific |
Cat #: F530L |
Superscript III Reverse Transcriptase |
Life Technologies |
Cat #: 18080-044 |
RNase H |
NEB |
Cat #: M0297L |
TURBO DNase (2 U/μL) |
Thermo Fisher Scientific |
Cat #: AM2238 |
KAPA HiFi hot start Taq |
Kapa Biosystems |
Cat #: kk2502 |
Dynabeads MyOne Streptavidin C1 |
Life Technologies |
Cat #: 65002 |
Cytiva Sera-Mag SpeedBeads Carboxyl Magnetic Beads, hydrophobic |
Fisher Scientific |
Cat #: 09-981-123 |
DSG (disuccinimidyl glutarate) |
Thermo Fisher Scientific |
Cat #: 20593 |
Proteinase K, Molecular Biology Grade |
NEB |
Cat #: P8107S |
RNase Inhibitor, Murine |
NEB |
Cat #: M0314L |
T4 Polynucleotide Kinase |
NEB |
Cat #: M0201L |
FastAP Thermosensitive Alkaline Phosphatase |
Thermo Fisher Scientific |
Cat #: EF0654 |
Protease Inhibitor Cocktail |
Promega |
Cat #: G6521 |
ProTEV Plus |
Promega |
Cat #: V6101 |
Vectashield |
Vector Labs |
Cat #: H-1000 |
DAPI |
Thermo Fisher Scientific |
Cat #: D1306 |
SpCas9 2NLS Nuclease (1000 pmol) |
Synthego |
N/A |
DMSO |
Sigma-Aldrich |
Cat #: D2650 |
DMEM/F-12, HEPES, no phenol red |
Life Technologies |
Cat #: 11039021 |
Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix |
Life Technologies |
Cat #: A1413202 |
N2 Supplement (100X) |
Thermo Fisher Scientific |
Cat #: 17502048 |
B27 Supplement (50X), minus Vitamin A |
Thermo Fisher Scientific |
Cat #:12587010 |
Animal Free Human Activin-A(e.coli) |
PeproTech |
Cat #:AF-120-14E |
Recombinant Human FGF-basic (FGF) |
PeproTech |
Cat #: 100-18B |
Recombinant Human EGF Protein, CF |
RnD (Perseus Proteomics) |
Cat #: 2028-EG-200 |
Mouse LIF |
Homemade |
N/A |
PD 0325901 |
Fisher Scientific |
Cat #: 4192 |
CHIR99021 |
Stemgent |
Cat #: 04-0004 |
Neurobasal Medium |
Life Technologies |
Cat #: 21103-049 |
DMEM: F12 |
Life Technologies |
Cat #: 11320-082 |
Knockout DMEM |
Life Technologies |
Cat #: 10829018 |
DMEM |
Life Technologies |
Cat #: 11995073 |
FBS |
Thermo Fisher Scientific |
Cat #: 10437028 |
Glutamax I |
Life Technologies |
Cat #: 35050061 |
MEM NEAA |
Life Technologies |
Cat #: 11140-050 |
20x Penicillin/Streptomycin |
Life Technologies |
Cat #: 15140-163 |
Gelatin from porcine skin, Type A |
Sigma-Aldrich |
Cat #: G2500 |
Accutase (cell dissociation) |
Life Technologies |
Cat #: A11105-01 |
Trypsin |
Life Technologies |
Cat #: 25200114 |
DPBS |
Fisher Scientific |
Cat #: SH3002802 |
UltraPure BSA (50 mg/mL) |
Thermo Fisher Scientific |
Cat #: AM2616 |
Bovine Serum Albumin (BSA) |
Sigma-Aldrich |
Cat #: A7906 |
Fish Skin Gelatin |
Sigma-Aldrich |
Cat #: G7765 |
Triton X-100 |
Sigma-Aldrich |
Cat #: T8787 |
Tween 20 |
Sigma-Aldrich |
Cat #: P9416 |
20X SSC |
Life Technologies |
Cat #:AM9765 |
PBS (10X), pH 7.4 |
Life Technologies |
Cat #: 70011069 |
Dextran sulphate sodium salt |
Sigma-Aldrich |
Cat #: D8906 |
Formamide |
Fisher Scientific |
Cat #: F84-1 |
Omnipur deionized formamide |
VWR |
Cat #: EM-4610 |
Lipofectamine 3000 |
Life Technologies |
Cat #: L3000015 |
RNAseOUT |
Life Technologies |
Cat #: 10777019 |
Opti-MEM I Reduced Serum Medium |
Thermo Fisher Scientific |
Cat #: 31985070 |
Tri Reagent |
Zymo Research |
Cat #: R2050-1-200 |
Thermo Scientific Pierce Methanol free Formaldehyde Ampules |
Thermo Fisher Scientific |
Cat #: 28908 |
Ribonucleoside Vanadyl Complex |
NEB |
Cat #: S1402S |
Actinomycin D |
Sigma-Aldrich |
Cat #: A9415 |
Indole-3-Acetic Acid |
Cayman Chemical Company |
Cat #: 16954 |
Glycine,bioultra, for molecular biology, ≥99.0% (NT) |
Sigma-Aldrich |
Cat #: 50046 |
Critical commercial assays
|
TrueSeq Stranded mRNA Library Prep Kit |
Illumina |
Cat #: 20020594 |
Chromium single cell 3’ reagent kit V3.1 |
10xGenomics |
Cat #: PN-1000121 |
Click-IT EdU Cell Proliferation Kit for Imaging |
Thermo Fisher Scientific |
Cat #: C10337
|
In-Fusion HD Cloning |
Clontech |
Cat #: 639649 |
BioPrime Array CGH Labeling System |
Life Technologies |
Cat #: 18095011 |
Quick Ligation Kit |
NEB |
Cat #: M2200L |
NEBNext Ultra End Repair/dA-tailing |
NEB |
Cat #: E7442L |
4D-NucleofectorTM X Kit |
Lonza |
Cat #: V4XP-3024 |
P3 Primary Cell 4D-Nucleofector Kit S |
Lonza |
Cat #: V4XP-3032 |
GeneJET Plasmid Miniprep Kit |
Thermo Fisher Scientific |
Cat #: FERK0503 |
Direct-zol RNA MiniPrep Kit with TRI-Reagent, Zymo-Spin IIC Columns |
Zymo Research |
Cat #: R2051 |
MinElute Gel Extraction Kit |
Qiagen |
Cat #: 28606 |
NucleoBond Xtra BAC |
Clontech |
Cat #: 740436.25 |
NucleoBond Xtra Maxi |
Clontech |
Cat #: 740414.50 |
Deposited data
|
Bulk RNA-seq, scRNA-seq, CLAP-seq, RAP-seq |
This study |
GSE181236 |
mus musculus Cas genome sequence |
EMBL-EBI |
ERP000042 |
mus musculus 129 genome sequence |
EMBL-EBI |
SRA: SRX037820 |
Hi-C and RNA-seq allelic counts in Smchd1−/− female NPCs |
(Wang et al., 2018) |
GSE99991 |
RNA-seq from SPOC-Bgl tethering to Xist |
(Dossin et al., 2020) |
GSE131784 |
Experimental models: Cell lines
|
Mouse ESCs 129S4/SvJae/castaneus F1 2-1 |
(Panning et al., 1997) |
N/A |
XistMS2-GFP ESCs (F1 2-1-XIST24MS2/MCP-GFP) |
This study |
N/A |
XistMS2-GFP/R26CIZ1mCherry |
This study |
N/A |
XistMS2-GFP/R26CIZ1Halo |
This study |
N/A |
XistMS2-GFP/R26CELF1mCherry |
This study |
N/A |
XistMS2-GFP/R26PCGF5Halo |
This study |
N/A |
XistMS2-GFP/R26PTBP1 Halo |
This study |
N/A |
XistMS2-GFP/PyP-CAG-HaloSPEN |
This study |
N/A |
XistMS2-GFP/PyP-CAG-HaloΔIDRSPEN |
This study |
N/A |
XistMS2-GFP/PyP-CAG-HaloΔRRMSPEN |
This study |
N/A |
Mouse ESCs 129S4/SvJae/castaneus F1 2-1-XIST12MS2 |
(Jonkers et al., 2008) |
N/A |
129S4ΔB/SvJae/castaneus F1 2-1 ESCs-XIST12MS2 |
This study |
N/A |
129S4ΔB/SvJae/castaneus F1 2-1 ESCs-XIST12MS2-R26SPENHalo |
This study |
N/A |
pSM33 tetO-Xist V6.5 male mouse ESCs |
(Engreitz et al., 2013) |
N/A |
pSM9 tetO-XistΔB V6.5 male mouse ESCs |
This study |
N/A |
36.11 tetO-Xist cDNA transgene chr 11, male mouse ESCs |
(Wutz and Jaenisch, 2000) |
N/A |
36.11 tetO-Xist cDNA transgene chr 11, male mouse ESCs- R26SPENHalo |
This study |
N/A |
Mouse ESCs TX1072-Spen-GFP/Spen-GFP-BglXist-mCherry |
(Dossin et al., 2020) |
N/A |
TX1072 ESCs -Spen-Halo/Spen-Halo |
(Dossin et al., 2020) |
N/A |
TX1072 ESCs-Spen-GFP/Spen-GFP |
(Dossin et al., 2020) |
N/A |
TX1072 ESCs-Spen-AID-GFP/Spen-AID-GFP |
(Dossin et al., 2020) |
N/A |
TX1072 ESCs -ΔIDRSpen-GFP/ΔIDRSpen-GFP |
This study |
N/A |
TX1072 ESCs-Spen-AID-GFP/Spen-AID-GFP-R26Spen-Halo |
This study |
N/A |
TX1072 ESCs-Spen-AID-GFP/Spen-AID-GFP-R26ΔIDRSpen-Halo |
This study |
N/A |
C127I |
ATCC |
Cat #: CRL-1616 |
Human fibroblasts - Abnormal Xi-Chromosome deletion |
Coriell |
Cat #: GM3827 |
Human fibroblasts - Abnormal Xi-Turner Syndrome |
Coriell |
Cat #: GM00735 |
Human fibroblasts - Abnormal Xi-Dicentric chromosome |
Coriell |
Cat #: GM06960 |
Human fibroblasts - Abnormal Xi-Dicentric chromosome |
Coriell |
Cat #: GM07213 |
Experimental models: Organisms/strains
|
DR4 mice (for feeders) |
The Jackson Laboratory |
Cat #: 003208 |
Oligonucleotides
|
Fluorescently labelled oligonucleotides used in oligoFISH, probes for RAP-seq and gRNAs for the IDRs deletion of SPEN |
This study |
See Table S2
|
Recombinant DNA
|
pMS2-GFP |
(Fusco et al., 2003) |
Addgene plasmid cat #: #27121 |
pCR4-24XMS2SL-stable |
(Bertrand et al., 1998) |
Addgene plasmid cat #: 31865 |
pBglII5k plasmid |
(Jonkers et al., 2008) |
N/A |
pBglII5k-24xMS2 plasmid |
This study |
N/A |
pBS31 (pgkATGfrt) plasmid |
(Beard et al., 2006) |
N/A |
pBS32 plasmid |
(Minkovsky et al., 2014) |
N/A |
pBS32-MCP-CIZ1 plasmid |
(Pandya-Jones et al., 2020) |
N/A |
FRT-neo plasmid |
(Beard et al., 2006) |
N/A |
I3-01-ct60GFP plasmid |
(Hsia et al., 2016) |
N/A |
pBS32-cage-60GFP plasmid |
This study |
N/A |
MXS_PGK::rtTA3-bGHpA |
(Sladitschek and Neveu, 2015) |
Addgene plasmid cat #: 62446 |
pBS31-MCP-GFP-rtTA3 plasmid |
This study |
N/A |
H2B-mCherry |
(Nam and Benezra, 2009) |
Addgene plasmid cat #: 20972 |
EasyFusion Halo-mAID |
(Gu et al., 2018a) |
Addgene plasmid cat #: 112852 |
R26-SA-EGFP-puro |
(Blelloch et al., 2004) |
Addgene plasmid cat #: 26890 |
pYM215-R26-SA/SD-puro |
This study |
N/A |
R26-CELF1-mCherry-puro plasmid |
This study |
N/A |
R26-PCGF5-Halo-puro plasmid |
This study |
N/A |
R26-PTBP1-Halo-puro plasmid |
This study |
N/A |
R26-CIZ1-Halo-puro plasmid |
This study |
N/A |
R26-CIZ1-mCherry-puro plasmid |
This study |
N/A |
R26-H2B-Halo-puro plasmid |
This study |
N/A |
pYM300-R26-Halo-SPEN-hygro plasmid |
This study |
N/A |
pYM301-R26-Halo-ΔIDR-SPEN-hygro plasmid |
This study |
N/A |
PyPP-CAG-Halo-full-length-Spen-V5 plasmid |
This study |
N/A |
PyPP-CAG-Halo-Spen-ΔIDR-V5 plasmid |
This study |
N/A |
PyPP-CAG-Halo-Spen-ΔRRM-V5 plasmid |
This study |
N/A |
full-length mSpen Entry Clone (Sp22) |
Alexander Shiskin |
N/A |
pFD46-R26-SPEN-hygro plasmid |
(Dossin et al., 2020) |
N/A |
pFD82-Cas9D10A-gRNA1 plasmid |
(Dossin et al., 2020) |
N/A |
pFD83-Cas9D10A-gRNA2 plasmid |
(Dossin et al., 2020) |
N/A |
p15A-31-17.9kb Xist plasmid |
(Pandya-Jones et al., 2020) |
N/A |
pCMV-Xist-PA |
(Wutz and Jaenisch, 2000) |
Addgene plasmid cat #: 26760 |
p13-5-Xist-Bdel plasmid |
This study |
N/A |
pPGK-Cre-bpA |
Klaus Rajewsky |
Addgene plasmid cat #: 11543 |
FlpO plasmid |
(Kranz et al., 2010) |
N/A |
BAC plasmid used to generate AtrX probe |
BacPac Consortium at Children’s Hospital Oakland Research Institute |
Cat #: RP23-265D6 |
Fosmid plasmid used to generate Mecp2 probe |
BacPac Consortium at Children’s Hospital Oakland Research Institute |
Cat #: WI-894A5 |
Fosmid plasmid used to generate Rlim probe |
BacPac Consortium at Children’s Hospital Oakland Research Institute |
Cat #: WI1-2704K12 |
BAC plasmid used to generate multispectral X chromosome barcoding |
BacPac Consortium at Children’s Hospital Oakland Research Institute |
Cat #: RP23-53H15 |
BAC plasmid used to generate multispectral X chromosome barcoding |
BacPac Consortium at Children’s Hospital Oakland Research Institute |
Cat #: RP23-83J1 |
BAC plasmid used to generate multispectral X chromosome barcoding |
BacPac Consortium at Children’s Hospital Oakland Research Institute |
Cat #: RP23-451D5 |
BAC plasmid used to generate multispectral X chromosome barcoding |
BacPac Consortium at Children’s Hospital Oakland Research Institute |
Cat #: RP24-81K23 |
BAC plasmid used to generate multispectral X chromosome barcoding |
BacPac Consortium at Children’s Hospital Oakland Research Institute |
Cat #: RP24-374B8 |
BAC plasmid used to generate multispectral X chromosome barcoding |
BacPac Consortium at Children’s Hospital Oakland Research Institute |
Cat #: RP23-401G5 |
BAC plasmid used to generate multispectral X chromosome barcoding |
BacPac Consortium at Children’s Hospital Oakland Research Institute |
Cat #: RP23-104K18 |
BAC plasmid to generate the Xist intron 1 probe |
BacPac Consortium at Children’s Hospital Oakland Research Institute |
Cat #: RP23-223G18 |
Software and algorithms
|
Fiji |
(Schindelin et al., 2012) |
https://fiji.sc/
|
ImageJ |
(Rueden et al., 2017) |
https://imagej.nih.gov/ij/
|
TrackMate |
(Tinevez et al., 2017) |
https://imagej.net/plugins/trackmate/
|
3D ImageJ Suite |
(Ollion et al., 2013) |
https://imagej.net/plugins/3d-imagej-suite/
|
Python |
(van Rossum and Drake, 2009) |
https://www.python.org/
|
Google Colaboratory |
Google Research |
https://research.google.com/colaboratory
|
PyTrackmate |
Hadrien Mary |
https://github.com/hadim/pytrackmate
|
pandas |
(The pandas development team, 2020) |
https://pandas.pydata.org/
|
NumPy |
(Harris et al., 2020) |
https://numpy.org/
|
SciPy |
(Virtanen et al., 2020) |
https://www.scipy.org/index.html
|
Matplotlib |
(Hunter, 2007) |
https://matplotlib.org/
|
Seaborn |
(Waskom, 2021) |
https://seaborn.pydata.org/index.html
|
Benchling |
The Benchling Life Sciences R&D |
https://benchling.com. |
MACS2 |
(Zhang et al., 2008) |
https://github.com/taoliu/MACS/
|
Bowtie2 |
(Langmead et al., 2009) |
http://bowtie-bio.sourceforge.net/bowtie2/index.shtml
|
TrimGalore (v0.4.1) |
Babraham Bioinformatics |
https://github.com/FelixKrueger/TrimGalore
|
bedtools (2.26.0) |
(Quinlan and Hall, 2010) |
http://bedtools.readthedocs.io/en/latest
|
samtools (v1.7) |
(Danecek et al., 2021) |
http://www.htslib.org/
|
bcftools |
Wellcome Sanger Institute |
http://www.htslib.org/
|
Picard (v2.1.0) |
The Broad Institute |
https://broadinstitute.github.io/picard/
|
Plyranges (v1.4.4) |
(Lee et al., 2019) |
https://bioconductor.org/packages/release/bioc/html/plyranges.html
|
Cellranger (v5.0.1) |
10xGenomics |
https://support.10xgenomics.com/single-cell-vdj/software/pipelines/latest/installation
|
Vartrix (v1.1.14) |
10xGenomics |
https://github.com/10XGenomics/vartrix/releases
|
STAR (v2.7.1a) |
(Dobin et al., 2013) |
https://github.com/alexdobin/STAR
|
GATK (v4.1.4.1) |
(Van der Auwera and O’Connor, 2020) |
https://gatk.broadinstitute.org/hc/en-us
|
R Software Package (v3.6) |
(R Core Team, 2021) |
https://www.r-project.org/
|
RStudio |
(RStudio Team, 2020) |
https://www.rstudio.com/
|
Tidyverse |
(Wickham et al., 2019) |
https://www.tidyverse.org/
|
ggpubr |
CRAN |
https://cran.r-project.org/web/packages/ggpubr/index.html
|
Deeptools |
(Ramirez et al., 2016) |
https://deeptools.readthedocs.io/en/3.4.3/index.html
|
ChIPpeakAnno |
(Zhu et al., 2010) |
http://bioconductor.org/packages/release/bioc/html/ChIPpeakAnno.html
|
EnrichedHeatmap |
(Gu et al., 2018b) |
https://bioconductor.org/packages/release/bioc/html/EnrichedHeatmap.html
|
MATLAB |
Mathworks |
https://www.mathworks.com/products/matlab.html
|
Mathematica (v10.1) |
Wolfram Research, Inc. |
https://www.wolfram.com/mathematica
|
Other
|
Salmon Sperm DNA |
Sigma-Aldrich |
Cat #: D9156 |
XMP X Green (mmX probe) |
MetaSystems Probes |
Cat #: D-1420-050-FI |
Mouse Cot1 DNA |
Life Technologies |
Cat #: 18440016 |
Mouse flow sorted chromosome X |
Gift form I. Solovei |
N/A |
high precision coverslips 12 mm round |
Azer Scientific |
Cat #: ES0117520 |
Correlative microscopy coverslips |
Ted Pella |
Cat #: 260511 |
Fixogum rubber cement |
Fisher Scientific |
Cat #: 11FIXO0125 |
μ-Slide 8 Well Glass Bottom |
ibidi |
Cat #: 80827 |
μ-Slide 4-well Glass Bottom |
ibidi |
Cat #: 80427 |
CoverGrip Coverslip Sealant |
VWR |
Cat #: 89411-108 |
Gene Pulser/MicroPulser Electroporation Cuvettes, 0.4 cm gap |
Bio-rad |
Cat #: 1652088 |
TetraSpeck Microspheres, 0.1 μm, fluorescent blue/green/orange/dark red |
Thermo Fisher Scientific |
Cat #: T7279 |