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| Status |
Public on Sep 05, 2017 |
| Title |
mESC_Inp_rep2_UNT |
| Sample type |
SRA |
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| Source name |
Mouse embryonic stem cells with Drosophila S2 line (SG4) spike-in
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| Organism |
Mus musculus |
| Characteristics |
cell line: CFP1 fl/fl (CFP1 conditional)
chip antibody: none
replicate: 2
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| Treatment protocol |
CFP1 fl/fl ESCs were treated with 4-hydroxytamoxifen (TAM) for 96 h to ablate CFP1 protein levels.
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| Growth protocol |
Mouse embryonic stem cells containing conditional alleles for CFP1 (CFP1 fl/fl) were grown on gelatin-coated plates in DMEM (Gibco, Carlsbad, CA) supplemented with 15 % FBS, 10 ng/mL leukemia-inhibitory factor, penicillin/streptomycin, beta-mercaptoethanol, L-glutamine and non-essential amino-acids. Drosophila S2 (SG4) cells were grown adhesively at 25°C in Schneider Drosophila Medium (Gibco), supplemented with 1x penicillin-streptomycin solution and 10% fetal bovine serum (Biosera) that was previously heat-inactivated at 60°C for 30 min.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP sequencing for H3K4me3 in mouse ES cells was performed by calibrated native ChIP sequencing. Calibrated ChIP-seq was carried out in biological triplicate for each cell line and condition. This was achieved by adding 2.5x10^6 Drosophila S2 (SG4) cells to 10x10^6 untreated or tamoxifen treated Cfp1 fl/fl cells. Nuclei were released by resuspending the mixed cell mixture in ice cold lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40). Nuclei were then washed, and resuspended in 1 ml (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40, 0.25 M sucrose, 3 mM CaCl2, 1x protease inhibitors, and incubated with 100U of MNase (Fermentas) at 37°C for 5 min followed by the addition of EDTA to halt the digestion. The supernatant was collected following centrifugation at 1500 g for 5 min at 4°C. The remaining pellet was incubated with 300 ul of nucleosome release buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.2 mM EDTA, 1x PIC) at 4°C for 1 h, then passed through a 27G needle using a 1 ml syringe, and spun at 1500 g for 5 min at 4°C. The two supernatants were combined. For each immunoprecipitation 20 µl of IPA300 agarose beads (RepliGen) were blocked with 1 mg/ml BSA and 1 mg/ml yeast tRNA in native ChIP incubation buffer (70 mM NaCl, 10 mM Tris-HCl pH 7.5, 2 mM MgCl2, 2 mM EDTA, 0.1% Triton X-100). Native chromatin was then diluted 10-fold in (70 mM NaCl, 10 mM Tris-HCl pH 7.5, 2 mM MgCl2, 2 mM EDTA, 0.1% TritonX-100, 1x PIC) and incubated with 20 µl of blocked beads per 1 ml of diluted chromatin at 4°C for 1 h. Beads were washed four times with native ChIP wash buffer (20 mM Tris-HCl pH 7.5, 2 mM EDTA, 125 mM NaCl, 0.1% Triton-X100), and once with 1 ml ice cold TE buffer. DNA was purified with the ChIP DNA Clean & Concentrator kit (Zymo Research). For CFP1 and T7-SET1A ChIP-seq in ES cells, an equal number of untreated and treated cells were resuspended in 10 mL of PBS and subjected to crosslink with 1% methanol-free formaldehyde (ThermoFisher) for 10 min at 25°C and quenched with 150 mM glycine for 10 min at RT. For T7-SET1A ChIP, cells were first crosslinked in 2 uM disuccinimidyl glutarate (DSG, ThermoFisher) at 25° C for 50 mins and then 1% formaldehyde was added for 10 min at 25°C. Cells were lysed on ice for 10 min in 1 mL of lysis buffer (50 mM HEPES pH7.9, 300 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.5% NP-40, 0.1% Na-deoxycholate, 0.1% SDS, 1x Complete EDTA-free inhibitor cocktail, 1 mM AEBSF) and then sonicated with Bioruptor Pico (Diagenode) for 20 min with 30s ON/OFF cycles (for double crosslinked chromatin, cells were sonicated for 23 min). Lysates were centrifuged for 10 min at 16000 g and the cleared chromatin was recovered and DNA quantified. For each IP, 300 ug of chromatin was diluted to 1 mL of lysis buffer and pre-cleared for 1 h at 4°C with IPA 300 resin beads (Repligen) blocked with yeast tRNA and BSA. Antibodies were then added to each IP and incubated overnight, followed by isolation of antibody-chromatin complexes with 40 uL of 50% blocked slurry beads per IP. Immunoprecipitates were washed 1x with lysis buffer, 1x with lysis buffer at 500 mM NaCl, 1x DOC buffer (10 mM Tris-HCl pH 8, 250 mM LiCl, 1 mM EDTA, 0.5% NP40, 0.5% Na-deoxycholate), 2x with TE pH 8. Chromatin was then eluted in 200 uL of elution buffer (1% SDS, 0.1 M NaHCO3) for 30 min at 30°C with vigorous shaking. Eluates and inputs were treated with DNase-free RNase (ThermoFisher) and crosslinks were reversed at 65 ˚C overnight with 200 mM NaCl and ProteinaseK solution (Sigma). DNA was purified with the ChIP DNA Clean & Concentrator kit (Zymo Research). For massively parallel sequencing, DNAs were post-sonicated with Bioruptor Pico (Diagenode) to a DNA fragment size of 200-300 bp as determined by Bioanalyser analysis.
Libraries were prepared with NEBNext Ultra DNA Library Prep Kit or with NEBNext EndPrep Dual Index for H3K4me3 ChIP-seq and quantified by qPCR using KAPA Illumina DNA standards as reference. Libraries were sequenced on an Illumina NextSeq500 platform.
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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 mouse mm10 genome using bowtie2 (Langmead and Salzberg, 2012) with the “—no-mixed” and “—no-discordant” options. Non-uniquely mapping reads were discarded. PCR duplicates were removed using SAMtools (Li et al., 2009). Biological replicates were randomly downsampled to contain the same number of reads for each individual replicate and merged when an internal calibration control was not available. ChIP-seq experiments which contained a spike-in genome (D. melanogaster) were aligned against a concatenated genome of the two genome sequences (mm10+dm6) using bowtie2 with the "--no-mixed" and "--no-discordant" options, and reads which mapped more than once were discarded. PCR duplicates were removed using samtools.
Spiking an identical concentration of D. melanogaster cells into our quantitative ChIP-seq experiments allows for calibration of each sample for IP efficiency and total mouse cell number. When comparing ChIP-seq for untreated and tamoxifen-treated cell lines using an internal calibration, the number of mm10 reads were randomly downsampled to reflect the total number of dm6 reads in that sample. Furthermore, to adjust for variation in the dm6/mm10 ratio between biological replicates, each sample was adjusted using the percentage of dm6 reads relative to mm10 reads in each sample's input DNA.
Calibrated native H3K4me3 ChIP-seq tracks were generated using DANPOS2 (Chen et al., 2013), whereas CFP1 and T7 ChIP-seq tracks were made using the pileup function of MACS2 (Zhang et al., 2008).
Genome_build: mm10
Supplementary_files_format_and_content: Bigwig files representing genome coverage for merged biological replicates.
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| Submission date |
Jan 12, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Vincenzo Di Cerbo |
| E-mail(s) |
[email protected]
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| Organization name |
University of Oxford
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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 |
GPL19057 |
| Series (2) |
| GSE93536 |
CFP1/SET1 complex occupancy and H3K4me3 profile in CFP1 fl/fl ES cells and rescue lines. [mESC_ChIPseq] |
| GSE93538 |
CFP1 engages in multivalent interactions with CpG island chromatin to recruit the SET1 complex and regulate gene expression. |
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| Relations |
| BioSample |
SAMN06219988 |
| SRA |
SRX2486178 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data not applicable for this record |
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