Human immunodeficiency virus-1 induces host genomic R-loops and preferentially integrates its genome near the R-loop regions
- Center for RNA Research, Institute for Basic Science, Republic of Korea
- School of Biological Sciences, Seoul National University, Republic of Korea
- Bioinformatics Institute, Seoul National University, Republic of Korea
- BK21 FOUR Intelligence Computing, Seoul National University, Republic of Korea
- Department of Computer Science and Engineering, Seoul National University, Republic of Korea
- SNU Institute for Virus Research, Seoul National University, Republic of Korea
Figures
Figure 1 with 6 supplements
HIV-1 infection induces genomic R-loop accumulation in cells at early post-infection.
(A) Summary of experimental design for DNA-RNA immunoprecipitation followed by cDNA conversion coupled with high-throughput sequencing (DRIPc-seq) in HeLa cells, primary CD4+ T cells, and Jurkat …
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Figure 1—source data 1
Original dot blots for Figure 1D, indicate the relevant dots and treatments.
- https://cdn.elifesciences.org/articles/97348/elife-97348-fig1-data1-v1.zip
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Figure 1—source data 2
Original files for dot blot analysis are displayed in Figure 1D.
- https://cdn.elifesciences.org/articles/97348/elife-97348-fig1-data2-v1.zip
Figure 1—figure supplement 1
Primary CD4+ T cells sorting strategies and GFP-HIV-1 infection.
(A) Gating strategy used to determine the efficiency of CD4+ T cells sorting from human PBMC. Pre-sorted PBMCs were staining with FITC-conjugated anti-CD4 and subjected fto positive CD4+ T cell …
Figure 1—figure supplement 2
Chromosomal position and DNA-RNA immunoprecipitation followed by cDNA conversion coupled with high-throughput sequencing (DRIPc-seq) signal for referenced R-loop-positive and –negative regions in HIV-1 infected HeLa cells.
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Figure 1—figure supplement 2—source data 1
- https://cdn.elifesciences.org/articles/97348/elife-97348-fig1-figsupp2-data1-v1.xlsx
Figure 1—figure supplement 3
Chromosomal position and DNA-RNA immunoprecipitation followed by cDNA conversion coupled with high-throughput sequencing (DRIPc-seq) signal for referenced R-loop-positive and –negative regions in HIV-1 infected primary CD4+ T cells.
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Figure 1—figure supplement 3—source data 1
- https://cdn.elifesciences.org/articles/97348/elife-97348-fig1-figsupp3-data1-v1.xlsx
Figure 1—figure supplement 4
Chromosomal position and DNA-RNA immunoprecipitation followed by cDNA conversion coupled with high-throughput sequencing (DRIPc-seq) signal for referenced R-loop-positive and –negative regions in HIV-1 infected Jurkat cells.
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Figure 1—figure supplement 4—source data 1
- https://cdn.elifesciences.org/articles/97348/elife-97348-fig1-figsupp4-data1-v1.xlsx
Figure 1—figure supplement 5
Genome browser screenshot over the HIV-1-induced R-loop forming positive or negative genomic regions.
(A–C), Genome browser screenshot over the P1 (A), P2 (B), and P3 (C) HIV-1 induced R-loop-positive chromosomal regions showing results from DNA-RNA immunoprecipitation followed by cDNA conversion …
Figure 1—figure supplement 6
Host cellular R-loop induction by HIV-1 infection is host-genome specific.
(A) DRIP followed by real-time polymerase chain reaction (DRIP-qPCR) using the anti-S 9.6 antibody at P1, P2, P3, N1, and N2 in HIV-1-infected cells with multiplicity of infection (MOI) of 0.6 …
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Figure 1—figure supplement 6—source data 1
Original dot blots for Figure 1—figure supplement 6B, indicating the relevant dots and treatments.
- https://cdn.elifesciences.org/articles/97348/elife-97348-fig1-figsupp6-data1-v1.zip
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Figure 1—figure supplement 6—source data 2
Original files for dot blot analysis are displayed in Figure 1—figure supplement 6B.
- https://cdn.elifesciences.org/articles/97348/elife-97348-fig1-figsupp6-data2-v1.zip
Figure 2 with 2 supplements
HIV-1-induced R-loops are enriched at both transcriptionally active and silent regions.
(A) Distribution of DNA-RNA immunoprecipitation followed by cDNA conversion coupled with high-throughput sequencing (DRIPc-seq) peak lengths for HIV-1-infected HeLa cells, primary CD4+ T cells, and …
Figure 2—figure supplement 1
R-loop induction by HIV-1 infection does not follow transcriptome changes in HeLa cells.
(A) Line graphs and heat maps representing expression levels of indicated repetitive elements (SINE, right; LINE, middle; LTR, left) at the indicated hpi of HIV-1 in HeLa cells. Data are presented …
Figure 2—figure supplement 2
RNA-seq analysis of relative gene expression levels of P1-3 and N1,2 R-loop regions.
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Figure 2—figure supplement 2—source data 1
- https://cdn.elifesciences.org/articles/97348/elife-97348-fig2-figsupp2-data1-v1.xlsx
Figure 3 with 1 supplement
R-loop inducible cell line model directly addresses R-loop-mediated HIV-1 integration site selection.
(A) Summary of the experimental design for R-loop inducible cell lines, pgR-poor and pgR-rich. (B) Gene expression of ECFP (gray) and mAIRN (red), as measured using RT-qPCR in pgR-poor or pgR-rich …
Figure 3—figure supplement 1
PiggyBac transposon-transposase insertion of R-loop forming and non-R-loop forming sequences in HeLa cells.
(A) Copy the number of piggyBac transposon inserts in each cell line constructed by transfecting the transposon vector and transposase-expressing vector. (B and C) Fold induction of gene expression …
Figure 4
HIV-1 prefers host genomic R-loop regions for its viral cDNA integration.
(A) Bar graphs showing the quantified number of HIV-1 integration sites per Mb pairs in total regions of 30 kb windows centered on DNA-RNA immunoprecipitation followed by cDNA conversion coupled …
Figure 5 with 1 supplement
HIV-1 integrase proteins directly bind to host genomic R-loops.
(A) Representative gel images for electrophoretic mobility shift assay (EMSA) of Sso7d-tagged HIV-1-integrase (E152Q) with R-loop and dsDNA, 10 nM nucleic acid substrate was incubated with …
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Figure 5—source data 1
Original gel image for Figure 5A, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97348/elife-97348-fig5-data1-v1.zip
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Figure 5—source data 2
Original files for electrophoretic mobility shift assay (EMSA) analysis are displayed in Figure 5A.
- https://cdn.elifesciences.org/articles/97348/elife-97348-fig5-data2-v1.zip
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Figure 5—source data 3
Original western blots for Figure 5C, indicate the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97348/elife-97348-fig5-data3-v1.zip
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Figure 5—source data 4
Original files for western blot analysis are displayed in Figure 5C.
- https://cdn.elifesciences.org/articles/97348/elife-97348-fig5-data4-v1.zip
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Figure 5—source data 5
Original western blots for Figure 5D, indicate the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97348/elife-97348-fig5-data5-v1.zip
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Figure 5—source data 6
Original files for western blot analysis are displayed in Figure 5D.
- https://cdn.elifesciences.org/articles/97348/elife-97348-fig5-data6-v1.zip
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Figure 5—source data 7
Original western blots for Figure 5E, indicate the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97348/elife-97348-fig5-data7-v1.zip
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Figure 5—source data 8
Original files for western blot analysis are displayed in Figure 5E.
- https://cdn.elifesciences.org/articles/97348/elife-97348-fig5-data8-v1.zip
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Figure 5—source data 9
Original dot blots for Figure 5F, indicate the relevant dots and treatments.
- https://cdn.elifesciences.org/articles/97348/elife-97348-fig5-data9-v1.zip
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Figure 5—source data 10
Original files for dot blot analysis are displayed in Figure 5F.
- https://cdn.elifesciences.org/articles/97348/elife-97348-fig5-data10-v1.zip
Figure 5—figure supplement 1
HIV-1 integrase proteins directly bind to host genomic R-loops.
(A) Representative gel images for electrophoretic mobility shift assay (EMSA) of Sso7d-tagged HIV-1-integrase (E152Q) with different types of nucleic acids substrates (R:D+ssDNA and Hybrid). 100 nM …
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Figure 5—figure supplement 1—source data 1
Original dot blots for Figure 5—figure supplement 1B, indicating the relevant dots and treatments.
- https://cdn.elifesciences.org/articles/97348/elife-97348-fig5-figsupp1-data1-v1.zip
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Figure 5—figure supplement 1—source data 2
Original files for dot blot analysis are displayed in Figure 5—figure supplement 1B.
- https://cdn.elifesciences.org/articles/97348/elife-97348-fig5-figsupp1-data2-v1.zip
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Figure 5—figure supplement 1—source data 3
Original western blots for Figure 5—figure supplement 1D, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97348/elife-97348-fig5-figsupp1-data3-v1.zip
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Figure 5—figure supplement 1—source data 4
Original files for western blot analysis are displayed in Figure 5—figure supplement 1D.
- https://cdn.elifesciences.org/articles/97348/elife-97348-fig5-figsupp1-data4-v1.zip
Author response image 1
Representative flow cytometry histograms of VSV-G-pseudotyped HIV-1-EGFP-infected HeLa cells at an MOI of 1, harvested at 48 hpi.
The cells were treated with DMSO, the indicated doses of nevirapine (NVP) (A) or indicated doses of raltegravir (RAL) (B) for 24 h before infection.
Author response image 2
Genome browser screenshot over the chromosomal regions in 20-kb windows centered on human AIRN showing results from DRIPc-seq in the indicated HIV-1-infected cells (blue, 0 hpi; yellow, 3 hpi; green, 6 hpi; red, 12 hpi).
Author response image 3
Dot blot analysis of cellular R-loops in cells infected with different virus particles.
(A) Dot blot analysis of the R-loop in gDNA extracts from HIV-1 infected U2OS cells with MOI of 0.6 harvested at 6 hpi. The gDNA extracts were incubated with or without RNase H in vitro before …
Author response image 4
Gating strategy used to determine HIV-1-infectivity in HeLa cells at 48 hpi.
Cells were infected with a known p24 antigen content in the stock of the VSV-G-pseudotyped HIV-1-EGFP-virus. The percentages of GFP-positive cell population are indicated.
Author response image 5
Bar graph indicating DRIPc-seq peak counts for HIV-1-infected primary CD4+ T cells harvested at the indicated hours post infection (hpi).
Pre-immunoprecipitated samples were untreated (−) or treated (+) with RNase H, as indicated. Each dot corresponds to an individual data set from two biologically independent experiments.
Author response image 6
Bar graphs showing the quantified number of HIV-1 integration sites per Mb pair in total regions of 30-kb windows centered on DRIPc-seq peaks from HIV-1 infected HeLa cells and primary CD4+ T cells (magenta) or non-R-loop region in the cellular genome (gray).
Each dot corresponds to an individual data set from two biologically independent experiments.
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Sequence-based reagent | P1 Fwd | This paper | TTATAAGTCAGCCTCCAGGATCAA | |
| Sequence-based reagent | P1 Rev | This paper | TTCAGGTCTAGGCAGTCTGA | |
| Sequence-based reagent | P2 Fwd | This paper | GGACAGATGACAGGGTCGC | |
| Sequence-based reagent | P2 Rev | This paper | ATGAGGAAGACCCCCTCGG | |
| Sequence-based reagent | P3 Fwd | This paper | CTCTGTGTAACGCTGGTGCT | |
| Sequence-based reagent | P3 Rev | This paper | ACACGCTTCTGACCACTAAGG | |
| Sequence-based reagent | N1 Fwd | This paper | TTGGCCCTACTGAATGATTGGT | |
| Sequence-based reagent | N1 Rev | This paper | TTAAGGCATGCTCAGGCGA | |
| Sequence-based reagent | N2 Fwd | This paper | TGAGATTTCAGGTTCCATGATTTG | |
| Sequence-based reagent | N2 Rev | This paper | TGCTCAGTGTTCTAATTTCCCTGT | |
| Sequence-based reagent | β-actin Fwd | This paper | AGAGCTACGAGCTGCCTGAC | |
| Sequence-based reagent | β-actin Rev | This paper | AGCACTGTGTTGGCGTACAG | |
| Sequence-based reagent | SH49 (ECFP Fwd) | Hamperl et al., 2017 | TGGTTTGTCCAAACTCATCAA | |
| Sequence-based reagent | SH40 (mAIRN Fwd) | Hamperl et al., 2017; | CGAGAGAGGCTAAGGGTGAA | |
| Sequence-based reagent | SH21 (ECFP/mAIRN Rev) | Hamperl et al., 2017; Braun, 2018 | ACATGGTCCTGCTGGAGTTC | |
| Sequence-based reagent | RT-qPCR P1 (TOR1AIP2) Fwd | This paper | CCTTGGTCTTTCCCACTTGAGTG | |
| Sequence-based reagent | RT-qPCR P1 (TOR1AIP2) Rev | This paper | GCAGGGTTAAAACCAGCTACTCG | |
| Sequence-based reagent | RT-qPCR P2 (DVL1) Fwd | This paper | GCATAACCGACTCCACCATGTC | |
| Sequence-based reagent | RT-qPCR P2 (DVL1) Rev | This paper | GATGGAGCCAATGTAGATGCCG | |
| Sequence-based reagent | RT-qPCR P3 (PKN2) Fwd | This paper | GCATCACCAACACTAAGTCCACG | |
| Sequence-based reagent | RT-qPCR P3 (PKN2) Rev | This paper | GCTTTTGACCGTCCAGGGACAT | |
| Sequence-based reagent | RT-qPCR N2 (CDK5RAP1) Fwd | This paper | AGAGTGGAAGCAGCCGTGTGTT | |
| Sequence-based reagent | RT-qPCR N2 (CDK5RAP1) Rev | This paper | GATCTTCCTCCGTCTCACCACA | |
| Sequence-based reagent | PCR primer 1.0 P5 | Sanz and Chédin, 2019 | AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGA | |
| Sequence-based reagent | PCR primer 2.0 P7 | Sanz and Chédin, 2019 | CAAGCAGAAGACGGCATACGAGAT | |
| Sequence-based reagent | Index Adapter 1 | Illumina | HeLa 0hpi Input replicate 1; Jurkat 0hpi Input replicate 1 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCACGATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 2 | Illumina | HeLa 3hpi Input replicate 1; Jurkat 3hpi Input replicate 1 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGATGTATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 3 | Illumina | HeLa 6hpi Input replicate 1; Jurkat 6hpi Input replicate 1 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTAGGCATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 4 | Illumina | HeLa 12hpi Input replicate 1; Jurkat 12hpi Input replicate 1 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGACCAATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 5 | Illumina | HeLa 0hpi RNH-IP replicate 1; Jurkat 0hpi RNH-IP replicate 1 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACACAGTGATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 6 | Illumina | HeLa 3hpi RNH-IP replicate 1; Jurkat 3hpi RNH-IP replicate 1 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCCAATATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 7 | Illumina | HeLa 6hpi RNH-IP replicate 1; Jurkat 6hpi RNH-IP replicate 1 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACCAGATCATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 8 | Illumina | HeLa 12hpi RNH-IP replicate 1; Jurkat 12hpi RNH-IP replicate 1 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACACTTGAATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 9 | Illumina | HeLa 0hpi RNH +IP replicate 1; Jurkat 0hpi RNH +IP replicate 1 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACGATCAGATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 10 | Illumina | HeLa 3hpi RNH +IP replicate 1; Jurkat 3hpi RNH +IP replicate 1 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACTAGCTTATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 11 | Illumina | HeLa 6hpi RNH +IP replicate 1; Jurkat 6hpi RNH +IP replicate 1 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGCTACATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 12 | Illumina | HeLa 12hpi RNH +IP replicate 1; Jurkat 12hpi RNH +IP replicate 1 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTTGTAATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 13 | Illumina | Jurkat 0hpi Input replicate 2 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGTCAAATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 14 | Illumina | Jurkat 3hpi Input replicate 2 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACAGTTCCATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 15 | Illumina | Jurkat 6hpi Input replicate 2 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACATGTCAATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 16 | Illumina | Jurkat 12hpi Input replicate 2 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCGTCCATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 17 | Illumina | Jurkat 0hpi RNH-IP replicate 2 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTAGAGATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 18 | Illumina | Jurkat 3hpi RNH-IP replicate 2 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTCCGCATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 19 | Illumina | Jurkat 6hpi RNH-IP replicate 2 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTGAAAATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 20 | Illumina | Jurkat 12hpi RNH-IP replicate 2 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTGGCCATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 21 | Illumina | Jurkat 0hpi RNH +IP replicate 2 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACGTTTCGATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 22 | Illumina | Jurkat 3hpi RNH +IP replicate 2 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGTACGATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 23 | Illumina | Jurkat 6hpi RNH +IP replicate 2 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACGAGTGGATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 24 | Illumina | Jurkat 12hpi RNH +IP replicate 2 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGTAGCATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 25 | Illumina | CD4 0hpi Input donor 1 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACACTGATATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 26 | Illumina | CD4 3hpi Input donor 1 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACATGAGCATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 27 | Illumina | CD4 6hpi Input donor 1 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACATTCCTATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 28 | Illumina | HeLa 0hpi Input replicate 2; CD4 12hpi Input donor 1 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACCAAAAGATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 29 | Illumina | HeLa 3hpi Input replicate 2; CD4 0hpi RNH-IP donor 1 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACCAACTAATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 30 | Illumina | HeLa 6hpi Input replicate 2; CD4 3hpi RNH-IP donor 1 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACCACCGGATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 31 | Illumina | HeLa 12hpi Input replicate 2; CD4 6hpi RNH-IP donor 1 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACCACGATATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 32 | Illumina | HeLa 0hpi RNH-IP replicate 2; CD4 12hpi RNH-IP donor 1 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACCACTCAATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 33 | Illumina | HeLa 3hpi RNH-IP replicate 2; CD4 0hpi RNH +IP donor 1 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACCAGGCGATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 34 | Illumina | HeLa 6hpi RNH-IP replicate 2; CD4 3hpi RNH +IP donor 1 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACCATGGCATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 35 | Illumina | HeLa 12hpi RNH-IP replicate 2; CD4 6hpi RNH +IP donor 1 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACCATTTTATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 36 | Illumina | HeLa 0hpi RNH +IP replicate 2; CD4 12hpi RNH +IP donor 1 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACCCAACAATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 37 | Illumina | HeLa 3hpi RNH +IP replicate 2; CD4 0hpi Input donor 2 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGGAATATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 38 | Illumina | HeLa 6hpi RNH +IP replicate 2; CD4 3hpi Input donor 2 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTAGCTATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 39 | Illumina | HeLa 12hpi RNH +IP replicate 2; CD4 6hpi Input donor 2 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTATACATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 40 | Illumina | CD4 12hpi Input donor 2 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTCAGAATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 41 | Illumina | CD4 0hpi RNH-IP donor 2 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACGACGACATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 42 | Illumina | CD4 3hpi RNH-IP donor 2 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACTAATCGATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 43 | Illumina | CD4 6hpi RNH-IP donor 2 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACTACAGCATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 45 | Illumina | CD4 12hpi RNH-IP donor 2 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACTCATTCATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 46 | Illumina | CD4 0hpi RNH +IP donor 2 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACTCCCGAATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 47 | Illumina | CD4 3hpi RNH +IP donor 2 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACTCGAAGATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 48 | Illumina | CD4 6hpi RNH +IP donor 2 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACTCGGCAATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | Index Adapter 49 | Illumina | CD4 12hpi RNH +IP donor 2 | GATCGGAAGAGCACACGTCTGAACTCCAGTCACAACAACATCTCGTATGCCGTCTTCTGCTTG |
| Sequence-based reagent | AE5316 | Achuthan et al., 2018 | First round LTR primer | TGTGACTCTGGTAACTAGAGATCCCTC |
| Sequence-based reagent | AE6380 | Achuthan et al., 2018 | HeLa replicate 1 5dpi Linker short / HeLa replicate 1 pgR-poor DOX- Linker short / CD4 +donor 1 Linker short | TAGTCCCTTAAGCGGAG-NH2 |
| Sequence-based reagent | AE6381 | Achuthan et al., 2018 | HeLa replicate 1 5dpi Linker long / HeLa replicate 1 pgR-poor DOX- Linker long / CD4 +donor 1 Linker long | GTAATACGACTCACTATAGGGCCTCCGCTTAAGGGAC |
| Sequence-based reagent | AE6382 | Achuthan et al., 2018 | HeLa replicate 1 5dpi Linker primer / HeLa replicate 1 pgR-poor DOX- Linker primer / CD4 +donor 1 Linker primer | CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTGTAATACGACTCACTATAGGGC |
| Sequence-based reagent | AE6404 | Achuthan et al., 2018 | HeLa replicate 1 5dpi Second round LTR primer / HeLa replicate 1 pgR-poor DOX- Second round LTR primer / CD4 +donor 1 Second round LTR primer | AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCGATGTGAGATCCCTCAGACCCTTTTAGTCAG |
| Sequence-based reagent | AE6380 | Achuthan et al., 2018 | HeLa replicate 2 5dpi Linker short / HeLa replicate 2 pgR-poor DOX +Linker short | TAGTCCCTTAAGCGGAG-NH2 |
| Sequence-based reagent | AE6381 | Achuthan et al., 2018 | HeLa replicate 2 5dpi Linker long /HeLa replicate 2 pgR-poor DOX +Linker long | GTAATACGACTCACTATAGGGCCTCCGCTTAAGGGAC |
| Sequence-based reagent | AE6382 | Achuthan et al., 2018 | HeLa replicate 2 5dpi Linker primer / HeLa replicate 2 pgR-poor DOX +Linker prime | CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTGTAATACGACTCACTATAGGGC |
| Sequence-based reagent | AE6404-1 | This paper | HeLa replicate 2 5dpi Second round LTR primer / HeLa replicate 2 pgR-poor DOX +Second round LTR primer | AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTTAGGCGAGATCCCTCAGACCCTTTTAGTCAG |
| Sequence-based reagent | AE6386 | Achuthan et al., 2018 | HeLa replicate 1 pgR-rich DOX- Linker short / CD4 +donor 2 Linker short | TACTATGACGGTGACGC-NH2 |
| Sequence-based reagent | AE6387 | Achuthan et al., 2018 | HeLa replicate 1 pgR-rich DOX- Linker long / CD4 +donor 2 Linker long | GAGAATCCATGAGTATGCTCACGCGTCACCGTCATAG |
| Sequence-based reagent | AE6388 | Achuthan et al., 2018 | HeLa replicate 1 pgR-rich DOX- Linker primer / CD4 +donor 2 Linker primer | CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTGAGAATCCATGAGTATGCTCAC |
| Sequence-based reagent | AE6406 | Achuthan et al., 2018 | HeLa replicate 1 pgR-rich DOX- Second round LTR primer / CD4 +donor 2 Second round LTR primer | AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTACAGTGGAGATCCCTCAGACCCTTTTAGTCAG |
| Sequence-based reagent | AE6456 | Achuthan et al., 2018 | HeLa replicate 1 pgR-poor DOX +Linker short | TAGACTGACGCAGTCTG-NH2 |
| Sequence-based reagent | AE6457 | Achuthan et al., 2018 | HeLa replicate 1 pgR-poor DOX +Linker long | GACGTACATACTGATCGCATAGCAGACTGCGTCAGTC |
| Sequence-based reagent | AE6458 | Achuthan et al., 2018 | HeLa replicate 1 pgR-poor DOX +Linker primer | CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTGACGTACATACTGATCGCATAG |
| Sequence-based reagent | AE6405 | Achuthan et al., 2018 | HeLa replicate 1 pgR-poor DOX +Second round LTR primer | AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTGACCAGAGATCCCTCAGACCCTTTTAGTCAG |
| Sequence-based reagent | AE6386 | Achuthan et al., 2018 | HeLa replicate 2 pgR-rich DOX +Linker short | TACTATGACGGTGACGC-NH2 |
| Sequence-based reagent | AE6387 | Achuthan et al., 2018 | HeLa replicate 2 pgR-rich DOX +Linker long | GAGAATCCATGAGTATGCTCACGCGTCACCGTCATAG |
| Sequence-based reagent | AE6388 | Achuthan et al., 2018 | HeLa replicate 2 pgR-rich DOX +Linker primer | CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTGAGAATCCATGAGTATGCTCAC |
| Sequence-based reagent | AE6406-1 | This paper | HeLa replicate 2 pgR-rich DOX +Second round LTR primer | AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGCCAATGAGATCCCTCAGACCCTTTTAGTCAG |
| Sequence-based reagent | AE6456 | Achuthan et al., 2018 | HeLa replicate 3 pgR-rich DOX- Linker short | TAGACTGACGCAGTCTG-NH2 |
| Sequence-based reagent | AE6457 | Achuthan et al., 2018 | HeLa replicate 3 pgR-rich DOX- Linker long | GACGTACATACTGATCGCATAGCAGACTGCGTCAGTC |
| Sequence-based reagent | AE6458 | Achuthan et al., 2018 | HeLa replicate 3 pgR-rich DOX- Linker primer | CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTGACGTACATACTGATCGCATAG |
| Sequence-based reagent | AE6411 | Achuthan et al., 2018 | HeLa replicate 3 pgR-rich DOX- Second round LTR primer | AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTAGTTCCGAGATCCCTCAGACCCTTTTAGTCAG |
| Sequence-based reagent | R-loop oligo1* | Kang et al., 2021 | R-loop; R:D+ssDNA | 5’-[Cy3]-GCC AGG GAC GAG GTG AAC CTG CAG GTG GGC GGC TAC TAC TTA GAT GTC ATC CGA GGC TTA TTG GTA GAA TTC GGC AGC GTC ATG C GA CGG C-3’ |
| Sequence-based reagent | R-loop oligo2* | Kang et al., 2021 | R-loop; dsDNA | 5’-GCC GTC GCA TGA CGC TGC CGA ATT CTA CCA CGC GAT TCA TAC CTG TCG TGC CAG CTG CTT TGC CCA CCT GCA GGT TCA CCT CGT CCC TGG C-3’ |
| Sequence-based reagent | R-loop RNA | Kang et al., 2021 | R-loop; R:D+ssDNA | 5’-[Cy5]-GCA GCU GGC ACG ACA GGU AUG AAU C-3’ |
| Sequence-based reagent | Homoduplex | Kang et al., 2021 | dsDNA | 5’-[Cy3]-GCC AGG GAC GAG GTG AAC CTG CAG GTG GGC AAA GCA GCT GGC ACG ACA GGT ATG AAT CGC GTG GTA GAA TTC GGC AGC GTC ATG CGA CGG C-3’ |
| Sequence-based reagent | Hybrid DNA | Kang et al., 2021 | Hybrid | 5’-CCC ATA CCG TAT AAC CAT TTG GCT GTC CAA GCT CCG GGT-3’ |
| Sequence-based reagent | Hybrid RNA | Kang et al., 2021 | Hybrid | 5’-[Cy5]-ACC CGG AGC UU G GAC AGC CAA AUG GU U AUA CGG UAU GGG-3’ |
| Sequence-based reagent | oligo 5 | Kang et al., 2021 | R:D+ssDNA | 5′GCAGTAGCATGACGCTGCTGAATTCTACCACGCTATGCT CTCGTCTAGGTTCACTCCGT CCCTGCGATTCATACCTGTCGTGCCAGCTGC |
| Other | Jurkat integration site | Li et al., 2020b | SRR12322252 | Source data |
| Other | TSA-seq_SPAD | Chen et al., 2018 | SRR3538917, SRR3538918, SRR3538919, SRR3538920 | Source data |
| Other | SPIN (Spatial Position Inference of the Nuclear genome) annotation of speckle | Wang et al., 2020 | https://github.com/ma-compbio/SPIN; ma-compbio, 2020 | Source data |
| Other | LADs | Meuleman et al., 2013 | GSE22428 | Source data |
Author response table 1
DRIPc-seq peak length and Chi-square p-value in CD4+ T cells from individual donor 1 and 2.
| Total DRIPc-seq peak length (replicate 1, bp) | Total DRIPc-seq peak length (replicate 2, bp) | Total DRIPc-seq peak length (overalp, bp) | Chi-square p-value | |
|---|---|---|---|---|
| Ohpi | 1910609 | 4722514 | 540640 | p < 1e-308 |
| 3hpi | 3630859 | 3308270 | 835457 | p < 1e-308 |
| 6hpi | 4195887 | 8496266 | 1485568 | p < 1e-308 |
| 12hpi | 4323824 | 13714402 | 1903473 | p < 1e-308 |
