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| Status |
Public on Apr 25, 2019 |
| Title |
PCGF6fl_TAM_NatInput_rep3 |
| Sample type |
SRA |
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| Source name |
Mouse embryonic stem cells with Drosophila spike-in, tamoxifen-treated (72hr), gDNA-seq
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| Organisms |
Drosophila melanogaster; Mus musculus |
| Characteristics |
cell line: Pcgf6fl/fl
replicate: 3
treatment agent: tamoxifen (OHT)
treatment time point: 72 hr
spike-in reference organism: Drosophila melanogaster
spike-in cell line: SG4
ChIP: none
antibody: none
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| Treatment protocol |
To induce conditional removal of distinct PRC1 complexes or their combinations in Pcgf4-/-; Pcgf2fl/fl, Pcgf1fl/fl, Pcgf3fl/fl, Pcgf5fl/fl, Pcgf6fl/fl, Pcgf3/5fl/fl, Pcgf1/3/5fl/fl, Pcgf1/3/5/2fl/fl, Pcgf1/3/5/6fl/fl and Ring1a-/-; Ring1bfl/fl ESC lines, cells were treated with 800 nM 4-hydroxytamoxifen (TAM) for 72 hours. To induce expression of Xist transgene, 129S1 x CAST ESCs were treated with 1.5 μg/mL doxycycline for 72 hours.
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| Growth protocol |
Mouse embryonic stem cells were grown on gelatin-coated plates at 37°C and 5% CO2, in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 15% fetal bovine serum (Labtech), 2 mM L-glutamine (Life Technologies), 1x penicillin-streptomycin solution (Life Technologies), 1x non-essential amino acids (Life Technologies), 0.5 mM beta-mercaptoethanol (Life Technologies), and 10 ng/mL leukemia inhibitory factor. Pcgf6fl/fl ESCs and Mus domesticus (129S1) x Mus castaneus F1 hybrid ESCs were grown on a monolayer of mitomycin-inactivated SNLP feeders (STO mouse fibroblasts expressing Neomycin, Puromycin resistance and Lif genes) in otherwise the same conditions as other mouse ESCs. Prior to harvesting these ES cells, the feeders were depleted by pre-plating trypsinised cells for 30 mins at 37°C on plates not coated with gelatin and discarding attached cells. Human HEK293T cells used for calibrated ChIP-seq were grown at 37°C and 5% CO2, in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (Labtech), 2 mM L-glutamine (Life Technologies), 1x penicillin-streptomycin solution (Life Technologies), and 0.5 Mm beta-mercaptoethanol (Life Technologies). Drosophila S2 (SG4) cells were grown adhesively at 25°C in Schneider’s Drosophila Medium (Life Technologies), supplemented with 1x penicillin-streptomycin solution (Life Technologies) and 10% heat-inactivated fetal bovine serum (Labtech).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For RING1B, SUZ12, PCGF1, PCGF2, CBX7 and PHC1 cChIP-seq, 5 x 10^7 mouse ESCs (both untreated and following 72 hours tamoxifen treatment) were mixed with 2 x 10^6 human HEK293T cells. Cells were resuspended in 10 ml phosphate buffered saline (PBS) and crosslinked at 25°C firstly with 2 mM DSG (Thermo Scientific) for 45 mins, and then with 1% formaldehyde (methanol-free, Thermo Scientific) for a further 15 minutes. Reactions were quenched by the addition of 125 mM glycine. Crosslinked cells were incubated in lysis buffer (50 mM HEPES pH 7.9, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton-X100) for 10 min at 4˚C. The released nuclei were then washed (10 mM Tris-HCl pH 8, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) 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% Na deoxycholate, 0.5% N-lauroylsarcosine) and sonicated for 30 min using a BioRuptor Pico sonicator (Diagenode), shearing genomic DNA to an average size of approximately 0.5 kb. Following sonication, TritonX-100 was added to a final concentration of 1%. For ChIP, sonicated chromatin was diluted 10-fold in ChIP dilution buffer (1% Triton-X100, 1 mM EDTA, 20 mM Tris-HCl pH 8, 150 mM NaCl) and pre-cleared for 1 hour using Protein A agarose beads (Repligen) blocked with 1 mg/ml BSA and 1 mg/ml yeast tRNA. For each ChIP reaction, 1ml of diluted and pre-cleared chromatin was incubated overnight with the appropriate antibody, anti-RING1B (CST D22F2, 3 ul), anti-SUZ12 (CST D39F6, 3 ul), anti-PCGF1 (in-house, 3 ul), anti-PCGF2 (sc-10744, 3 ul), anti-Cbx7 (ab21873, 4 ul), anti-Phc1 (CST 1F3F3, 6 ul). Antibody-bound chromatin was captured using blocked protein A agarose for 2 hours at 4°C and collected by centrifugation. 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-links were reversed overnight at 65°C with 200 mM NaCl and 2 µl of RNase A (Sigma). A matched input sample (corresponding to 10% of original ChIP reaction) was identically treated. The following day ChIP samples and Inputs were incubated with Proteinase K (Sigma) for 1.5 hours at 56°C and purified using ChIP DNA Clean and Concentrator Kit (Zymo Research). For H2AK119ub1 and H3K27me3 native cChIP-seq, 5 x 10^7 mouse ESCs (both untreated and following 72 hours tamoxifen treatment) were mixed with 2 x 10^7 Drosophila SG4 cells in PBS. Mixed cells were pelleted and nuclei were released by resuspending in ice cold lysis buffer (10mM Tris-HCl pH 8.0, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40, 5 mM N-ethylmaleimide). Nuclei were then washed, and resuspended in 1 ml of MNase digestion buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40, 0.25M sucrose, 3mM CaCl2, 10 mM N-ethylmaleimide, 1x protease inhibitors (Sigma)). Each sample was then incubated with 200 units of MNase (Fermentas) at 37°C for 5 min, followed by the addition of 4 mM EDTA to halt MNase digestion. Following centrifugation at 1500 g for 5 min at 4°C, the supernatant (S1) was retained. 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, 1x protease inhibitors, 10 mM N-ethylmaleimide) at 4°C for 1 h, passed five times through a 27G needle using a 1 ml syringe, and spun at 1500 g for 5 min at 4°C. The second supernatant (S2) was collected and combined with corresponding S1 sample from above. A small amount of S1/S2 DNA was purified and visualized on a 1.5% agarose gel to confirm digestion to mostly mono-nucleosomes. For ChIP experiments, S1/S2 nucleosomes were diluted 10-fold in native ChIP incubation buffer (70 mM NaCl, 10 mM Tris pH 7.5, 2 mM MgCl2, 2 mM EDTA, 0.1% Triton, 1x protease inhibitor cocktail (Roche), 10 mM N-ethylmaleimide (NEM)), and 1 ml aliquots were made. Each ChIP reaction was then incubated overnight at 4°C with 5 ul of anti-H2AK119ub1 (CST D27C4) or anti-H3K27me3 (in-house). Antibody-bound nucleosomes were captured using protein A agarose (Repligen) beads, pre-blocked in native ChIP incubation buffer supplemented with 1 mg/ml BSA and 1 mg/ml yeast tRNA, for 1 hour at 4°C and collected by centrifugation. Immunoprecipitated material was washed four times with Native ChIP wash buffer (20 mM Tris pH 7.5, 2 mM EDTA, 125 mM NaCl, 0.1% Triton-X100) and once with Tris-EDTA buffer (10 mM Tris pH 8, 1 mM EDTA). ChIP DNA was eluted using 100 ul of elution buffer (1% SDS, 0.1 M NaHCO3), and then purified using ChIP DNA Clean and Concentrator Kit (Zymo Research). For each individual ChIP sample, DNA from a matched Input control (corresponding to 10% of original ChIP reaction) was also purified.
Libraries for both ChIP and Input samples were prepared using NEBNext Ultra DNA Library Prep Kit for Illumina, 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 Illumina NextSeq 500 platform in biological triplicate unless otherwise specified. For allele-specific analysis in 129S1 x CAST hybrid ESC line with inducible fill-length Xist transgene, native cChIP-seq libraries were sequenced as 80 bp paired-end reads on Illumina NextSeq 500 platform to increase the number of reads overlapping allele-specific SNPs.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Paired-end reads were aligned to the genome sequence of concatenated mouse and spike-in genomes (mm10+dm6 for native cChIP-seq and mm10+hg19 for cross-linked cChIP-seq) using Bowtie 2 with the “--no-mixed” and “--no-discordant” options specified. Reads that were mapped more than once were discarded, followed by removal of PCR duplicates with SAMTools for native cChIP-seq or Sambamba for cross-linked cChIP-seq.
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 annotation of genomic intervals and data visualisation, mm10 reads were randomly subsampled using factors that reflected the total number of dm6 (or hg19) reads in each sample. To account for any variation in spike-in cell mixing in different biological replicates, the downsampling factors were additionally corrected using the ratio of dm6 (or hg19)/mm10 total read counts in corresponding Input samples.
For each condition, biological replicates were merged for downstream applications. Genome coverage tracks were generated using the pileup function from MACS2 (Zhang et al. 2008).
RING1B and SUZ12 peak sets were generated with MACS2 (--broad) using corresponding Input samples for background normalisation. For each factor, a set of peaks identified in all biological replicates (intersection) was manually curated to remove sequencing artefacts. Classical Polycomb chromatin domains were identified by k-means clustering of ±10 kb windows centred on RING1B peaks based on their RING1B and SUZ12 chromatin occupancy using deeptools (v.3.0.1). This generated three clusters, two of which displayed high enrichment of both RING1B and SUZ12 and were combined into one set of genomic intervals.
Allele-specific analysis of H2AK119ub1 cChIP-seq was performed as described previously (Pintacuda et al. 2017). Briefly, paired-end reads were aligned using STAR (with “--outFilterMultimapNmax 1”, “--outFilterMismatchNmax 2”, “--alignEndsType EndToEnd” parameters) against the concatenated mm10+dm6 genome, in which polymorphic SNPs for 129S1 and CAST mouse strains were N-masked. Reads that were mapped more than once were discarded and PCR duplicates were removed with Sambamba. Reads mapping to mm10 genome were randomly subsampled using downsampling factors calculated based on spike-in calibration as described previously. To separate reads specifically mapping to the 129S1 and CAST alleles, we used SNPsplit (Krueger et al. 2016) with the paired-end mode.
Genome_build: mm10
Supplementary_files_format_and_content: bigWig files representing genome coverage for merged replicates of spike-in normalised cChIP-seq and Input profiles
Supplementary_files_format_and_content: bed files with enrichment peaks for RING1B and SUZ12 generated with MACS2 (--broad) from at least three biological replicates (intersection)
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| Submission date |
Sep 06, 2018 |
| Last update date |
Apr 25, 2019 |
| Contact name |
Nadezda A Fursova |
| E-mail(s) |
nfursova.msu@gmail.com
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| Organization name |
University of Oxford
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| Department |
Department of Biochemistry
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| Lab |
Klose lab
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| Street address |
South Parks Rd
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| City |
Oxford |
| ZIP/Postal code |
OX13QU |
| Country |
United Kingdom |
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| Platform ID |
GPL25537 |
| Series (2) |
| GSE119618 |
Synergy between variant PRC1 complexes defines Polycomb-mediated gene repression (ChIP-Seq) |
| GSE119620 |
Synergy between variant PRC1 complexes defines Polycomb-mediated gene repression |
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| Relations |
| BioSample |
SAMN09986652 |
| SRA |
SRX4650796 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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