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| Status |
Public on Jul 19, 2021 |
| Title |
RING1AKO.RING1BAID_NativeInput_IAA_2h_rep3 |
| Sample type |
SRA |
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| Source name |
Mouse embryonic stem cells with Drosophila SG4 spike-in, auxin-treated (2h IAA)
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| Organisms |
Drosophila melanogaster; Mus musculus |
| Characteristics |
cell type: Mouse embryonic stem cells
genotype/variation: E14 Rosa26::Tir1; Ring1A-/-; AID-Ring1B
replicate: 3
treatment: auxin (IAA)
treatment duration: 2 hours
spike-in reference organism: Drosophila melanogaster
spike-in cell line: SG4
chip antibody: none
antibody info.: none
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| Treatment protocol |
To induce AID-RING1B degradation, water-dissolved auxin (indole-3-acetic acid (IAA), Sigma) was mixed with cell medium to the final concentration of 500 µM and added to PRC1deg cells at designated times before harvesting by trypsinisation. To induce dTAG-SUZ12 degradation, PRC2deg cells were treated with 100 nM dTAG-13 compound for 2 hours.
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| Growth protocol |
Mouse ESCs were grown on gelatin-coated plates, in Dulbecco’s Modified Eagle Medium (DMEM, Life Technologies) supplemented with 15% fetal bovine serum (FBS, Labtech), 0.5 mM beta-mercaptoethanol (Life Technologies), 2 mM L-glutamine (Life Technologies), 1x penicillin-streptomycin (Life Technologies), 1x non-essential amino acids (Life Technologies) and 10 ng/mL leukemia inhibitory factor. Cells were cultured at 37°C with 5% CO2. For calibration of genomic experiments, we used either Drosophila S2 (SG4) cells, cultured at 25°C in Schneider’s Drosophila Medium (Life Technologies), supplemented with 10% heat-inactivated FBS and 1x penicillin-streptomycin, or human HEK293T cells, cultured at 37°C with 5% CO2, in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 1x penicillin-streptomycin and 0.5 mM beta-mercaptoethanol.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For RING1B and SUZ12 cChIP-seq, 5x107 mouse ESCs were first crosslinked with 2 mM disuccinimidyl glutarate (DSG, Thermo Scientific) while rotating for 45 min at 25°C, and then with 1% formaldehyde (methanol-free, Thermo Scientific) for further 15 min. Reactions were quenched by addition of 125 mM glycine. Mouse ESCs were then mixed with 2x106 double-crosslinked HEK293T cells and incubated in lysis buffer (50 mM HEPES pH 7.9, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1x PIC) for 10 min at 4°C. The released nuclei were washed (10 mM Tris-HCl pH 8, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1x PIC) for 5 min at 4°C. Chromatin was then resuspended in 1 mL of sonication buffer (10 mM Tris-HCl pH 8, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroylsarcosine, 1x PIC) and sonicated for 30 min using the BioRuptor Pico (Diagenode). Following sonication, Triton X-100 was added to a final concentration of 1%. The supernatant was collected after centrifugation at 20,000 g for 10 min at 4°C. For ChIP, chromatin was diluted 10-fold in ChIP dilution buffer (1% Triton X-100, 1 mM EDTA, 20 mM Tris-HCl pH 8, 150 mM NaCl, 1x PIC) and pre-cleared for 1 h with either protein A agarose beads (Repligen, for RING1B ChIP) or protein A magnetic Dynabeads (Invitrogen, for SUZ12 ChIP) blocked with 1 mg/mL bovine serum albumin (BSA) and 1 mg/mL yeast tRNA. For each ChIP reaction, 1 mL of diluted and pre-cleared chromatin was incubated overnight with the appropriate antibody, either anti-RING1B (CST, D22F2, 3 μl), or anti-SUZ12 (CST, D39F6, 3 μl). Antibody-bound chromatin was captured using blocked protein A beads (agarose for RING1B, magnetic Dynabeads for SUZ12) for at least 2 h at 4°C and collected by centrifugation/on a magnetic rack. ChIP washes were performed as described previously (Farcas et al. 2013). ChIP DNA was eluted in elution buffer (1% SDS, 0.1 M NaHCO3) and cross-linking was reversed overnight at 65°C with 200 mM NaCl and 2 μL RNase A (Sigma). A matched input sample (1/10 of original ChIP reaction) was treated identically. The following day, ChIP samples and inputs were incubated with Proteinase K (Sigma) for 1.5 h at 56°C and purified using ChIP DNA Clean and Concentrator Kit (Zymo Research). For Pol II cChIP-seq, 5x107 mouse ESCs were crosslinked in 1% formaldehyde for 10 min at 25°C and then quenched with 125 mM glycine. Mouse ECSs were mixed with 2x106 single-crosslinked HEK293T cells and incubated in FA-lysis buffer (50 mM HEPES pH 7.9, 150 mM NaCl, 2 mM EDTA, 0.5 mM EGTA, 0.5% NP-40, 0.1% sodium deoxycholate, 0.1% SDS, 10 mM NaF, 1 mM AEBSF, 1x PIC) for 10 min. Chromatin was sonicated for 30 min using the BioRuptor Pico, centrifuged at 20,000 g for 10 min at 4°C, and supernatant taken as chromatin. For ChIP, 300 ug of chromatin was diluted to 1 ml in FA-lysis buffer and pre-cleared for 1 h with protein A agarose beads blocked with 1 mg/mL BSA and 1 mg/mL yeast tRNA. For each ChIP reaction, diluted and pre-cleared chromatin was incubated overnight with the appropriate antibody, anti-Rbp1-NTD (CST, D8L4Y, 15 μl) to detect total Pol II, anti-Rbp1-CTD-Ser5P (CST, D9N5I, 12.5 μl), or anti-Rbp1-CTD-Ser2P (CST, E1Z3G, 12.5 μl). Antibody-bound chromatin was isolated using blocked protein A agarose beads for 3 h at 4°C. Washes were performed with FA-Lysis buffer, FA-Lysis buffer containing 500 mM NaCl, DOC buffer (250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 2 mM EDTA, 10 mM Tris–HCl pH 8), followed by two washes with TE buffer pH 8. ChIP DNA was eluted, de-crosslinked and purified as described above, along with the matched input samples. For H2AK119ub1, H3K27me3 and H3K4me3 cChIP-seq, 5x107 mouse ESCs were mixed with 2x107 Drosophila SG4 cells in PBS. The cells were pelleted and nuclei were released by resuspending in ice-cold lysis buffer (10 mM Tris-HCl pH 8, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40, 5 mM NEM). Nuclei were then washed, and resuspended in 1 mL of MNase digestion buffer (10 mM Tris-HCl pH 8, 10 mM NaCl, 3 mM MgCl2, 0.25 M sucrose, 3 mM CaCl2, 10 mM NEM, 1x PIC). Samples were digested with 150 units of MNase (Fermentas) for 5 min at 37°C, stopped by 4 mM EDTA. The supernatant (S1) was collected following centrifugation at 1500 g for 5 min at 4°C. The remaining pellet was incubated with 300 μl of nucleosome release buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.2 mM EDTA, 10 mM NEM, 1x PIC) at 4°C for 1 h, passed five times through a 27G needle, and spun at 1500 g for 5 min at 4°C. The second supernatant (S2) was collected and combined with corresponding S1 supernatant, and stored at -80°C until use. Digestion to predominantly mono-nucleosomal fragments was confirmed by agarose gel electrophoresies of purified DNA. Native chromatin was diluted 10-fold in native ChIP incubation buffer (70 mM NaCl, 10 mM Tris–HCl pH 7.5, 2 mM MgCl2, 2 mM EDTA, 0.1% TritonX-100, 10 mM NEM, 1x PIC). For each ChIP reaction, 1 ml of diluted nucleosomes was incubated overnight at 4°C with the appropriate antibody, anti-H2AK119ub1 (CST, D27C4, 5 μL), anti-H3K27me3 (in-house, 5 μL) or anti-H3K4me3 (in-house, 3 μL). Antibody-bound nucleosomes were captured by incubation for 1 h at 4°C with protein A agarose beads, pre-blocked overnight in native ChIP incubation buffer supplemented with 1 mg/ml BSA and 1 mg/ml yeast tRNA. The beads were then washed four times with native ChIP wash buffer (20 mM Tris–HCl pH 7.5, 2 mM EDTA, 125 mM NaCl, 0.1% TritonX-100) and once with TE buffer pH 8. Immunoprecipitated DNA was eluted using 100 μl of elution buffer (1% SDS, 0.1 M NaHCO3) and purified using ChIP DNA Clean and Concentrator Kit. DNA from a matched input sample (corresponding to 10% of the original ChIP reaction) purified in the same way.
cChIP-seq libraries for both ChIP and input samples were prepared using NEBNext Ultra DNA Library Prep Kit for Illumina (double-crosslinked and native ChIP-seq) or NEBNext Ultra II DNA Library Prep Kit for Illumina (Pol II ChIP-seq), following manufacturer’s guidelines. Samples were indexed using NEBNext Multiplex Oligos. The average size and concentration of all libraries was analysed using the 2100 Bioanalyzer High Sensitivity DNA Kit (Agilent) followed by qPCR using SensiMix SYBR (Bioline, UK) and KAPA Illumina DNA standards (Roche). Libraries were sequenced as 40 bp paired-end reads on the Illumina NextSeq 500.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
RING1AKO_RING1BAID_Ninput_AUX_2h_rep5_S4
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| Data processing |
Paired-end reads were aligned to the concatenated mouse (mm10) and spike-in (dm6 for native, hg19 for cross-linked cChIP-seq) genome sequences using Bowtie 2 (“–no-mixed” and “–no-discordant” options). Only uniquely mapped reads were kept for downstream analysis, after removal of PCR duplicates with Sambamba.
To internally calibrate cChIP-seq experiments, we spiked-in a fixed number of control cells (Drosophila SG4 cells for native cChIP-seq and human HEK293T cells for cross-linked cChIP-seq) to each experimental sample
For visualization of cChIP-seq sequencing data and annotation of genomic regions with read counts, uniquely mapped mouse reads were normalized using dm6 or hg19 spike-in as described previously (Furosva et al., 2019). Briefly, mouse reads were randomly subsampled based on the total number of spike-in (dm6 or hg19) reads in each sample. Additionally, to account for possible minor variations in spike-in cell mixing between cChIP-seq replicates, we corrected the subsampling factors by using the ratio of spike-in to mouse total read counts in the corresponding input samples. Prior to merging normalized replicates for visualization and analysis, read coverage across regions of interest (RING1B peaks for RING1B, SUZ12, H2AK119ub1 and H3K27me3, or TSS ± 2.5 kb for total Pol II, Ser5P-Pol II and H3K4me3, or gene bodies for Ser2P) was analyzed using multiBamSummary and plotCorrelation functions from deepTools , confirming a high correlation between replicates (Pearson's correlation coefficient > 0.9). Genome coverage tracks for cChIP-seq were generated using the pileup function from MACS2.
To identify genomic regions bound by PRC1 we performed peak calling using MACS2 (“BAMPE” and “–broad” options) on the RING1B cChIP-seq data with corresponding input samples for background normalization. A set of peaks identified in all biological replicates of untreated PRC1deg cells was used for further analysis, after filtering out several peaks identified following 4 hours of auxin treatment (i.e. following loss of RING1B) and manually removing sequencing artefacts. In total, 7240 stringent RING1B peaks were identified.
Genome_build: mm10
Supplementary_files_format_and_content: bigWig files representing genome coverage for merged replicates of spike-in normalised cChIP-seq.
Supplementary_files_format_and_content: bed file for a set of enrichment peaks for RING1B
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| Submission date |
Oct 12, 2020 |
| Last update date |
Jul 19, 2021 |
| Contact name |
Paula Dobrinić |
| Organization name |
University of Oxford
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| Department |
Department of Biochemistry
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| Lab |
Rob Klose lab
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| Street address |
South Parks Road
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| City |
Oxford |
| ZIP/Postal code |
OX1 3QU |
| Country |
United Kingdom |
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| Platform ID |
GPL25537 |
| Series (2) |
| GSE159398 |
PRC1 drives Polycomb-mediated gene repression by controlling transcription initiation and burst frequency [ChIP-seq] |
| GSE159400 |
PRC1 drives Polycomb-mediated gene repression by controlling transcription initiation and burst frequency |
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| Relations |
| BioSample |
SAMN16422130 |
| SRA |
SRX9281768 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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