|
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 |
