RNA Polymerase II transcription independent of TBP in murine embryonic stem cells
- Department of Biochemistry and Molecular Biology, Life Sciences Institute, University of British Columbia, Canada
- Department of Pathology and Laboratory Medicine, University of British Columbia, Canada
- Michael Cuccione Childhood Cancer Research Program, BC Children’s Hospital Research Institute, Canada
Figures
Figure 1 with 3 supplements
Global Polymerase II (Pol II)-mediated transcription is TATA-box binding protein (TBP)-independent in mouse embryonic stem cells (mESCs).
(A) Schematic of indole-3-acetic acid (IAA)-mediated degradation of mAID-TBP. (B) Western blot analyses of whole cell lysates for control and IAA-treated C64 mESCs blotting for ɑ-TBP, with ɑ-Tubulin …
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Figure 1—source data 1
Full uncropped membrane scans of immunoblot analyses with α-TBP control and indole-3-acetic acid (IAA)-treated C64 whole cell lysates without and with annotations.
- https://cdn.elifesciences.org/articles/83810/elife-83810-fig1-data1-v2.zip
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Figure 1—source data 2
Full uncropped membrane scans of immunoblot analyses with α-Tubulin for control and indole-3-acetic acid (IAA)-treated C64 whole cell lysates without and with annotations.
- https://cdn.elifesciences.org/articles/83810/elife-83810-fig1-data2-v2.zip
Figure 1—figure supplement 1
Characterization of TATA-box binding protein (TBP) degradation.
(A) Cell growth curve of C64 mouse embryonic stem cells (mESCs) from 6 to 12 hr of treatment with DMSO (Con, black) or indole-3-acetic acid (IAA) (IAA, red). Error bars represent standard deviation; …
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Figure 1—figure supplement 1—source data 1
Full uncropped membrane scans of immunoblot analyses with α-TBP and α-H3.3 for control and indole-3-acetic acid (IAA)-treated C64 cell lysates (input), cytoplasmic fractions (cyto), and chromatin (pellet) without and with annotations.
Relevant bands for Figure 1—figure supplement 1C are highlighted with a red rectangle. L=ladder, x=lanes not used in Figure 1—figure supplement 1C.
- https://cdn.elifesciences.org/articles/83810/elife-83810-fig1-figsupp1-data1-v2.zip
Figure 1—figure supplement 2
Replicate analysis of Polymerase II (Pol II) cleavage under targets and tagmentation (CUT&Tag) and NET-seq data.
(A) Genome-wide average plots (top) and heatmaps (bottom) arranged by decreasing Pol II occupancy for ChIPseqSpikeInFree and Escherichia coli and S2 spike-in normalized Pol II CUT&Tag in a 2 kb …
Figure 1—figure supplement 3
TATA-box binding protein (TBP) is not required for transcription of specific subsets of genes.
(A) Average plots and heatmaps k-means clustering with k=3 of the log2 indole-3-acetic acid (IAA)/control of TBP cleavage under targets and tagmentation (CUT&Tag) data (left). Polymerase II (Pol II) …
Figure 2 with 1 supplement
TATA-box binding protein (TBP) is required for Pol III transcription of tRNAs in mouse embryonic stem cells (mESCs).
(A) Gene browser tracks of tRNA65-LeuCAA (top) and tRNA64-GlnCTG/tRNA1010-LeuCAA (bottom) for cleavage under targets and tagmentation (CUT&Tag) analyses of TBP (orange), Pol III (brown), and NET-seq …
Figure 2—figure supplement 1
Replicate analysis of TATA-box binding protein (TBP) and Pol III cleavage under targets and tagmentation (CUT&Tag).
(A) Western blot analyses for the Pol III subunit α-RPC7 and α-TBP of control and indole-3-acetic acid (IAA)-treated C64 mESCs. Tubulin is used as a loading control. (B) Gene browser tracks of tRNA65…
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Figure 2—figure supplement 1—source data 1
Full uncropped membrane scans of immunoblot analyses with α-RPC7 for control and indole-3-acetic acid (IAA)-treated C64 whole cell lysates without and with annotations.
Relevant bands for Figure 2—figure supplement 1A are highlighted with a red rectangle. L=ladder, x=lanes not used in Figure 2—figure supplement 1A.
- https://cdn.elifesciences.org/articles/83810/elife-83810-fig2-figsupp1-data1-v2.zip
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Figure 2—figure supplement 1—source data 2
Full uncropped membrane scans of immunoblot analyses with α-TBP control and indole-3-acetic acid (IAA)-treated C64 whole cell lysates without and with annotations.
Relevant bands for Figure 2—figure supplement 1A are highlighted with a red rectangle. L=ladder, x=lanes not used in Figure 2—figure supplement 1A.
- https://cdn.elifesciences.org/articles/83810/elife-83810-fig2-figsupp1-data2-v2.zip
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Figure 2—figure supplement 1—source data 3
Full uncropped membrane scans of immunoblot analyses with α-Tubulin control and indole-3-acetic acid (IAA)-treated C64 whole cell lysates without and with annotations.
Relevant bands for Figure 2—figure supplement 1A are highlighted with a red rectangle. L=ladder, x=lanes not used in Figure 2—figure supplement 1A.
- https://cdn.elifesciences.org/articles/83810/elife-83810-fig2-figsupp1-data3-v2.zip
Figure 3 with 1 supplement
TATA-box binding protein (TBP) is dispensable for gene activation by heat shock and retinoic acid differentiation in mouse embryonic stem cells (mESCs).
(A) Schematic of heat shock treatments for C64 mESCs. After 6 hr of treatment with auxin (indole-3-acetic acid [IAA]) or DMSO, cells were either collected or subjected to heat shock at 42°C for 30 …
Figure 3—figure supplement 1
Replicate analysis of TATA-box binding protein (TBP) and Polymerase II (Pol II) cleavage under targets and tagmentation (CUT&Tag), and NET-seq data on heat shock (HS)- and retinoic acid (RA)-induced genes.
(A) Gene browser tracks of Dnajb1 for CUT&Tag analyses of Pol II (green) and TBP (orange) in control, indole-3-acetic acid (IAA)-treated, HS and HS IAA-treated C64 mouse embryonic stem cells (mESCs) …
Figure 4 with 1 supplement
TATA-box binding protein (TBP) paralog TBP-related factor 2 (TRF2) is expressed in mouse embryonic stem cells (mESCs), but does not functionally replace TBP.
(A) Western blot analyses of whole cell lysates for α-TRF2 and α-H3K27me3 in control and indole-3-acetic acid (IAA)-treated C64 mESCs. (B) Western blot analyses of whole cell lysates for α-TRF2 and …
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Figure 4—source data 1
Full uncropped membrane scans of immunoblot analyses with α-TRF2 and α-H3K27me3 control and indole-3-acetic acid (IAA)-treated C64 whole cell lysates without and with annotations.
- https://cdn.elifesciences.org/articles/83810/elife-83810-fig4-data1-v2.zip
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Figure 4—source data 2
Full uncropped membrane scans of immunoblot analyses with α-HA control B8HA, B8, and C64 whole cell lysates without and with annotations.
- https://cdn.elifesciences.org/articles/83810/elife-83810-fig4-data2-v2.zip
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Figure 4—source data 3
Full uncropped membrane scans of immunoblot analyses with α-TRF2 control B8HA, B8, and C64 whole cell lysates without and with annotations.
- https://cdn.elifesciences.org/articles/83810/elife-83810-fig4-data3-v2.zip
Figure 4—figure supplement 1
Characterization of TRF2 knock-out B8 cells.
(A) Gene browser tracks of Tbpl1 (top) and Tbpl2 (bottom) for Polymerase II (Pol II) cleavage under targets and tagmentation (CUT&Tag) (green) and chrRNA-seq (blue) in C64 mouse embryonic stem cells …
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Figure 4—figure supplement 1—source data 1
Full uncropped membrane scans of immunoblot analyses with α-TRF2 control B8 and C64 whole cell lysates without and with annotations.
Relevant bands for Figure 4—figure supplement 1F are highlighted with a red rectangle. L=ladder, x=lanes not used in Figure 4—figure supplement 1F.
- https://cdn.elifesciences.org/articles/83810/elife-83810-fig4-figsupp1-data1-v2.zip
Figure 5 with 1 supplement
The TFIID complex forms upon TATA-box binding protein (TBP) depletion.
(A) Top: Western blot analyses for α-TAF4, α-TBP, and α-Tubulin of control and indole-3-acetic acid (IAA)-treated C64 whole cell extracts used in the TAF4 IP-MS analyses. Input represents 5% of the …
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Figure 5—source data 1
Full uncropped membrane scans of immunoblot analyses with α-TAF4 for control and indole-3-acetic acid (IAA)-treated C64 whole cell extracts without and with annotations.
- https://cdn.elifesciences.org/articles/83810/elife-83810-fig5-data1-v2.zip
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Figure 5—source data 2
Full uncropped membrane scans of immunoblot analyses with α-TBP for control and indole-3-acetic acid (IAA)-treated C64 whole cell extracts without and with annotations.
- https://cdn.elifesciences.org/articles/83810/elife-83810-fig5-data2-v2.zip
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Figure 5—source data 3
Full uncropped membrane scans of immunoblot analyses with α-Tubulin for control and indole-3-acetic acid (IAA)-treated C64 whole cell extracts without and with annotations.
- https://cdn.elifesciences.org/articles/83810/elife-83810-fig5-data3-v2.zip
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Figure 5—source data 4
Full uncropped membrane scans of immunoblot analyses with α-TAF4 for TAF4 IPs from control and indole-3-acetic acid (IAA)-treated C64 whole cell lysates without and with annotations.
- https://cdn.elifesciences.org/articles/83810/elife-83810-fig5-data4-v2.zip
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Figure 5—source data 5
Full uncropped membrane scans of immunoblot analyses with α-TBP for TAF4 IPs from control and indole-3-acetic acid (IAA)-treated C64 whole cell lysates without and with annotations.
- https://cdn.elifesciences.org/articles/83810/elife-83810-fig5-data5-v2.zip
Figure 5—figure supplement 1
TAF4 IP-MS analyses in control and TATA-box binding protein (TBP)-depleted cells.
(A) Volcano plot of the log2 fold change of proteins enriched in α-TAF4 IPover mouse IgG from control C64 mESCs, versus the -log10 transformed t-test p-value in control C64 mouse embryonic stem …
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Figure 5—figure supplement 1—source data 1
Full uncropped membrane scans of immunoblot analyses with α-TAF4 for gel filtration fractions 1–24 from control C64 cells without and with annotations.
Relevant bands for Figure 5—figure supplement 1C are highlighted with a red rectangle. L=ladder, in = input, x=lanes not used in Figure 5—figure supplement 1C.
- https://cdn.elifesciences.org/articles/83810/elife-83810-fig5-figsupp1-data1-v2.zip
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Figure 5—figure supplement 1—source data 2
Full uncropped membrane scans of immunoblot analyses with α-TAF4 for gel filtration fractions 25–47 from control C64 cells without and with annotations.
Relevant bands for Figure 5—figure supplement 1C are highlighted with a red rectangle. L=ladder, in = input, x=lanes not used in Figure 5—figure supplement 1C.
- https://cdn.elifesciences.org/articles/83810/elife-83810-fig5-figsupp1-data2-v2.zip
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Figure 5—figure supplement 1—source data 3
Full uncropped membrane scans of immunoblot analyses with α-TBP for gel filtration fractions 1–24 from control C64 cells without and with annotations.
Relevant bands for Figure 5—figure supplement 1C are highlighted with a red rectangle. L=ladder, in = input, x=lanes not used in Figure 5—figure supplement 1C.
- https://cdn.elifesciences.org/articles/83810/elife-83810-fig5-figsupp1-data3-v2.zip
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Figure 5—figure supplement 1—source data 4
Full uncropped membrane scans of immunoblot analyses with α-TBP for gel filtration fractions 25–47 from control C64 cells without and with annotations.
Relevant bands for Figure 5—figure supplement 1C are highlighted with a red rectangle. L=ladder, in = input, x=lanes not used in Figure 5—figure supplement 1C.
- https://cdn.elifesciences.org/articles/83810/elife-83810-fig5-figsupp1-data4-v2.zip
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Figure 5—figure supplement 1—source data 5
Full uncropped membrane scans of immunoblot analyses with α-TAF4 for gel filtration fractions 1–24 from indole-3-acetic acid (IAA)-treated C64 cells without and with annotations.
Relevant bands for Figure 5—figure supplement 1C are highlighted with a red rectangle. L=ladder, in = input, x=lanes not used in Figure 5—figure supplement 1C.
- https://cdn.elifesciences.org/articles/83810/elife-83810-fig5-figsupp1-data5-v2.zip
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Figure 5—figure supplement 1—source data 6
Full uncropped membrane scans of immunoblot analyses with α-TAF4 for gel filtration fractions 25–47 from indole-3-acetic acid (IAA)-treated C64 cells without and with annotations.
Relevant bands for Figure 5—figure supplement 1C are highlighted with a red rectangle. L=ladder, in = input, x=lanes not used in Figure 5—figure supplement 1C.
- https://cdn.elifesciences.org/articles/83810/elife-83810-fig5-figsupp1-data6-v2.zip
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Figure 5—figure supplement 1—source data 7
Full uncropped membrane scans of immunoblot analyses with α-TBP for gel filtration fractions 1–24 from indole-3-acetic acid (IAA)-treated C64 cells without and with annotations.
Relevant bands for Figure 5—figure supplement 1C are highlighted with a red rectangle. L=ladder, in = input, x=lanes not used in Figure 5—figure supplement 1C.
- https://cdn.elifesciences.org/articles/83810/elife-83810-fig5-figsupp1-data7-v2.zip
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Figure 5—figure supplement 1—source data 8
Full uncropped membrane scans of immunoblot analyses with α-TBP for gel filtration fractions 25–47 from indole-3-acetic acid (IAA)-treated C64 cells without and with annotations.
Relevant bands for Figure 5—figure supplement 1C are highlighted with a red rectangle. L=ladder, in = input, x=lanes not used in Figure 5—figure supplement 1C.
- https://cdn.elifesciences.org/articles/83810/elife-83810-fig5-figsupp1-data8-v2.zip
Figure 6 with 3 supplements
Effects of TATA-box binding protein (TBP) depletion on the DNA binding of TAF1, TAF4, and TFIIA.
(A) Gene browser tracks of Actb (top) and Gapdh (bottom) for cleavage under targets and tagmentation (CUT&Tag) analyses of TAF1 (blue), TAF4 (red), and TFIIA (gray). TBP signal (orange) from Figure 1…
Figure 6—figure supplement 1
Replicate analysis of Taf1 and Taf4 cleavage under targets and tagmentation (CUT&Tag), and NET-seq data.
(A) Gene browser tracks of Gapdh (left) and Actb (right) for CUT&Tag analyses of TAF1 (blue) and TAF4 (red) with four biological replicates of TAF1 and two biological replicates of TAF4 in control …
Figure 6—figure supplement 2
Replicate analysis of TFIIA cleavage under targets and tagmentation (CUT&Tag).
(A) Left: Boxplots showing abundance of TFIIA-γ from α-TAF4 IP-MS analyses of control (blue) and indole-3-acetic acid (IAA)-treated (red) C64 mouse embryonic stem cells (mESCs). Protein levels were …
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Figure 6—figure supplement 2—source data 1
Full uncropped membrane scans of immunoblot analyses with α-TBP for control and indole-3-acetic acid (IAA)-treated C64 whole cell lysates without and with annotations.
Relevant bands for Figure 6—figure supplement 2A are highlighted with a red rectangle. L=ladder, x=lanes not used in Figure 6—figure supplement 2A.
- https://cdn.elifesciences.org/articles/83810/elife-83810-fig6-figsupp2-data1-v2.zip
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Figure 6—figure supplement 2—source data 2
Full uncropped membrane scans of immunoblot analyses with α-TFIIA-γ for control and indole-3-acetic acid (IAA)-treated C64 whole cell lysates without and with annotations.
Relevant bands for Figure 6—figure supplement 2A are highlighted with a red rectangle. L=ladder, x=lanes not used in Figure 6—figure supplement 2A.
- https://cdn.elifesciences.org/articles/83810/elife-83810-fig6-figsupp2-data2-v2.zip
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Figure 6—figure supplement 2—source data 3
Full uncropped membrane scans of immunoblot analyses with α-Tubulin for control and indole-3-acetic acid (IAA)-treated C64 whole cell lysates without and with annotations.
Relevant bands for Figure 6—figure supplement 2A are highlighted with a red rectangle. L=ladder, x=lanes not used in Figure 6—figure supplement 2A.
- https://cdn.elifesciences.org/articles/83810/elife-83810-fig6-figsupp2-data3-v2.zip
Figure 6—figure supplement 3
Comparisons between Polymerase II (Pol II) TAF1, TAF4, and TFIIA.
(A) Genome-wide average plots and heatmaps of TAF1 (left), TAF4 (middle), and TFIIA (right) cleavage under targets and tagmentation (CUT&Tag) arranged by k-means k=3 log2 indole-3-acetic acid …
Author response image 1
Author response image 2
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Mus musculus, male) | JM8.N4 mouse ES cells | KOMP repository | RRID: CVCL_J962 | Parental cell line used for all genetic manipulations |
| Cell line (Mus musculus, male) | mAID-TBP KI C94 | https://doi.org/10.7554/eLife.35621 | C94 | Endogenous knock-in of the minimal auxin inducible degron to N-terminus of TBP in JM8.N4 cells |
| Cell line (Mus musculus, male) | Halo-Pol II C64 | https://doi.org/10.7554/eLife.35621 | C64 | Endogenous knock-in of the HaloTag to C-terminus of Rbp1, largest subunit of Pol II, in mAID-TBP C94 cells |
| Cell line (Mus musculus, male) | B8 | This paper | Endogenous knock-out of the Tbpl1 gene from Halo-Pol II C64 cells – see Materials and Methods | |
| Cell line (Mus musculus, male) | B8HA | This paper | Overexpressed HA-tagged TRF2 in B8 cells – see Materials and Methods | |
| Cell line (Drosophila melanogaster) | S2 | Gift from Dr. Eric Jan | Spike-in of 20% (by cell number) in CUT&Tag and qRT-PCR | |
| Transfected construct (Mus musculus) | pTRF2-HA | This paper | HA tagged TRF2 cDNA expressing plasmid – see Materials and Methods | |
| Transfected construct (Mus musculus) | pCas9-mCherry-TRF2-1 | This paper; backbone: cat #64324 (Addgene) | Targets Tbpl1 for knockout - see Materials and Methods | |
| Transfected construct (Mus musculus) | pCas9-mCherry-TRF2-2 | This paper; backbone: cat #64324 (Addgene) | Targets Tbpl1 for knockout - see Materials and Methods | |
| Antibody | α-TBP (Mouse, monoclonal) | Abcam | cat:ab51841 | Western blot (1:3000) ChIP (5 μg) CUT&Tag (1 μg) IF (1:100) |
| Antibody | α-Pol II (Rpb1 CTD) | Cell Signaling Technologies | cat:D1G3K | CUT&Tag (1 μg) |
| Antibody | α-H3K27me3 (Rabbit, monoclonal) | Cell Signaling Technologies | cat:C36B11 | Western blot (1:7000) CUT&Tag (1 μg) |
| Antibody | α-HA (Rabbit, polyclonal) | Epicypher | cat:13–2010 | Western blot (1:1000) CUT&Tag (1 μg) |
| Antibody | α-Tubulin (Rabbit, polyclonal) | Abcam | cat:ab6046 | Western blot (1:7000) |
| Antibody | α-Pol III RPC32 (mouse monoclonal) | Santa Cruz Biotechnology | cat:sc-48365 | Western blot (1:2000) |
| Antibody | α-TAF4 (mouse monoclonal) | Santa Cruz Biotechnology | cat:sc-136093 | Western blot (1:2000) CUT&Tag (1 μg) Co-IP (3 μg) |
| Antibody | α-H3.3 (mouse monoclonal) | Abnova | cat:H00003021-M01 | Western blot (1:5000) |
| Antibody | α-TRF2 (mouse monoclonal) | Gift from Dr. László Tora | Western blot (1:3000) | |
| Antibody | α-TFIIA-γ (mouse monoclonal) | Santa Cruz Biotechnology | cat:sc-374483 | Western blot (1:3000) |
| Antibody | α-TFIIA-γ (rabbit polyclonal) | Santa Cruz Biotechnology | cat:sc-25365 | CUT&Tag (1 μg) |
| Antibody | IRDye 800CW α-Mouse IgG (Goat polyclonal) | Licor | cat:926–32210 | Western blot (1:20000) |
| Antibody | IRDye 800CW α-Rabbit IgG (Goat polyclonal) | Licor | cat:925–32211 | Western blot (1:20000) |
| Antibody | IRDye 680RD α-Mouse IgG (Goat polyclonal) | Licor | cat:926–68070 | Western blot (1:20000) |
| Antibody | IRDye 680RD α-Rabbit IgG (Goat polyclonal) | Licor | cat:926–68701 | Western blot (1:20000) |
| Antibody | α-Mouse IgG (Rabbit, polyclonal) | Abcam | cat:ab46540 | ChIP (5 μg) CUT&Tag (1:100) |
| Antibody | α-TAF1 (Rabbit, monoclonal) | Cell Signaling Technologies | cat:D6J8B | CUT&Tag (1 μg) |
| Antibody | α-Rabbit IgG (Guinea Pig, polyclonal) | Antibodies-online | cat:ABIN101961 | CUT&Tag (1:100) |
| Antibody | Alexa Fluor 594 α-Mouse IgG (Goat, polyclonal) | Invitrogen | cat:A-11005 | IF (1:100) |
| Antibody | α-Mouse IgG (mouse, monoclonal) | Santa Cruz Biotechnology | cat:sc-2025 | Co-IP (3 μg) |
| Sequence-based reagent | Biotinylated oligonucleotides for NET-seq libraries | This paper | See Materials and Methods and Supplementary file 5 for full list | |
| Sequence-based reagent | qRT-PCR oligonucleotides | This paper | See Supplementary file 6 for full list | |
| Sequence-based reagent | ChIP-qPCR oligonucleotides | This paper | See Supplementary file 7 for full list | |
| Peptide, recombinant protein | pA-Tn5 | Gift from Dr. Steven Henikoff | Diluted to 1 x in each reaction | |
| Peptide, recombinant protein | pAG-Tn5 | Epicypher | cat:15–1017 | Diluted to 1 x in each reaction |
Additional files
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Supplementary file 1
Top upregulated/downregulated genes extracted from differential gene expression (DGE) analysis of polymerase II (Pol II) cleavage under targets and tagmentation (CUT&Tag) for all genes in control and IAA-treated C64 mESCs based on Figure 1F.
- https://cdn.elifesciences.org/articles/83810/elife-83810-supp1-v2.docx
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Supplementary file 2
Raw values of a few top upregulated HS genes extracted from differential gene expression (DGE) analysis of polymerase II (Pol II) cleavage under targets and tagmentation (CUT&Tag) on all genes in control vs. HS-treated C64 mESCs.
- https://cdn.elifesciences.org/articles/83810/elife-83810-supp2-v2.docx
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Supplementary file 3
Gene ontology analysis of the top upregulated genes extracted from differential gene expression (DGE) analysis of polymerase II (Pol II) cleavage under targets and tagmentation (CUT&Tag) in control vs. HS-treated C64 mESCs from Figure 1—figure supplement 3A.
- https://cdn.elifesciences.org/articles/83810/elife-83810-supp3-v2.docx
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Supplementary file 4
Raw values of a few top upregulated RA genes extracted from edgeR differential gene expression (DGE) analysis of polymerase II (Pol II) cleavage under targets and tagmentation (CUT&Tag) on all genes in control vs. RA-treated C64 mESCs.
- https://cdn.elifesciences.org/articles/83810/elife-83810-supp4-v2.docx
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Supplementary file 5
Biotinylated snRNA, snoRNA, lncRNA, and rRNA depletion oligonucleotides for NET-seq library construction.
Oligonucleotides were modified with the addition of a 5’ biotin group and purified by HPLC.
- https://cdn.elifesciences.org/articles/83810/elife-83810-supp5-v2.docx
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Supplementary file 6
Primers used to knock-out Tbpl1, confirm Tbpl1 knock-out, and to test Gapdh mRNA levels via qRT-PCR.
- https://cdn.elifesciences.org/articles/83810/elife-83810-supp6-v2.docx
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Supplementary file 7
Primers used to test TATA-box binding protein (TBP) depletion via ChIP-qPCR.
- https://cdn.elifesciences.org/articles/83810/elife-83810-supp7-v2.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/83810/elife-83810-mdarchecklist1-v2.docx
