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| Status |
Public on Nov 26, 2024 |
| Title |
PNUTSdTAG-SPT6-dTAG-rep3 |
| Sample type |
SRA |
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| Source name |
E14 PNUTS-dTAG
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| Organisms |
Homo sapiens; Mus musculus |
| Characteristics |
cell line: E14 PNUTS-dTAG
cell type: Mouse embryonic stem cells
spike-in cell_line: HEK293T
treatment: 100 nM dTAG13
treatment time: 2h
additional treatment: none
antibody: anti-SPT6 (Cell Signaling (D6J9H))
replicate: 3
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| Treatment protocol |
PNUTS-dTAG E14 mESCs were treated with 100 nM dTAG-13 (Tocris Bioscience) 2 hours to induce protein depletion. For rescue experiments in the E14 TetON-3G PNUTS-dTAG ESC line, DOX induction of the rescue protein (WT, W401A or TND-M) was started 2 hours prior dTAG treatment.
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| Growth protocol |
Mouse embryonic stem cells were grown in Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific) supplemented with fetal bovine serum (FBS, 10% Sigma), 1x non-essential amino acids (Thermo Fisher Scientific), 2 mM L-glutamine (Thermo Fisher Scientific), 1x penicillin/streptomycin (Thermo Fisher Scientific), 0.5 mM beta-mercaptoethanol (Thermo Fisher Scientific), and 10 ng/ml leukaemia inhibitory factor (produced in-house). ESCs were grown on gelatinised plates at 37⁰C and 5% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
5 x 10^7 ESCs were fixed with 1% formaldehyde (methanol-free, Thermo Fisher Scientific) for 10 min at 25⁰C. Fixation was quenched using glycine added to 125 mM. Cells were then pelleted at 1000xg for 5 min and washed with PBS. Cross-linked ESCs were mixed with 1 x 10^6 cross-linked HEK 293T(1% formaldehyde for 10 min). Chromatin was prepared by incubation in 1 ml FA-lysis buffer (50 mM HEPES pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.5% NP-40, 0.1% Na-deoxycholate, 0.1% SDS, 1x PIC, 10 mM NaF) on ice for 10 min. Chromatin was sonicated using a BioRuptor Pico sonicator (Diagenode) at 4⁰C for 23 cycles (30s on/off) to an average size of 0.5 kb. The sonicated material was pelleted at 20,000xg for 20 min, and the supernatant taken as sonicated chromatin. 300 μg chromatin was used per IP. Chromatin was diluted to 1 ml total volume per IP in FA-lysis buffer. An additional volume of diluted chromatin was taken to use as an input sample. Protein A agarose beads (Repligen) were blocked with 1 mg/ml BSA and 1 mg/ml yeast tRNA in 1x TE buffer at 4⁰C for 1 hr. Chromatin was pre-cleared with agarose beads (40 μl slurry beads per ChIP) at 4⁰C for 1 - 2 hr. The input sample was taken from the pre-cleared chromatin, and the remainder was immunoprecipitated overnight at 4⁰C with the respective antibody. Antibody-bound chromatin was precipitated for 3 hr at 4⁰C using 40 μl slurry of blocked Protein A agarose beads. Washes were carried out for 5 min each at 4⁰C, using ice-cold FA-lysis buffer, FA-lysis buffer with 500 mM NaCl, 1x DOC buffer (10 mM Tris HCl, pH 8, 250 mM LiCl, 1 mM EDTA , 0.5% NP-40, 0.5% Na-deoxycholate), and 2 washes with TE buffer. DNA was eluted in elution buffer (1% SDS and 0.1 M NaHCO3). Cross-links were reversed for ChIPs and inputs at 65⁰C overnight with 200 mM NaCl and 2 μl RNase A (Sigma). Samples were then incubated with 20 μg Proteinase K for 1 hr at 45⁰C. DNA for ChIPs and inputs was purified using a ChIP DNA Clean & Concentrator kit (Zymo Research). Purified DNA was analysed using ChIP-qPCR.
cChIP-seq libraries were prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina or NEBNext UltraExpress 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 were analysed using the 2100 Bioanalyzer High Sensitivity DNA Kit (Agilent) followed by quantification using Qubit HS DNA assay. Libraries were sequenced as 40 bp paired-end reads in at least biological triplicate on Illumina NextSeq 500 platform.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
SPT6 ChIP in PNUTS-dTAG mESCs treated with dTAG-13 to deplete PNUTS-dTAG
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| Data processing |
For cChIP-seq, paired-end reads were aligned to the concatenated mouse mm10 and spike-in genomes (mm10+hg19) using Bowtie2 with the ‘-no-mixed’ and ‘-no-discordant’ options. Reads that mapped more than once were discarded and PCR duplicates were removed using Sambamba.
Sequencing datasets were calibrated to the spike-in human genomes, as described previously (Fursova et al. 2019). For cChIP-seq, the number of mm10 reads were randomly downsampled to reflect the total number of hg19 reads in that sample. Furthermore, to adjust for any variation in cell mixing, each sample was adjusted using the percentage of dm6 reads relative to mm10 reads in the relevant input sample. After normalisation, read coverages for individual biological replicates were compared across regions of interest using the multiBamSummary and plotCorrelation functions from deepTools.
Assembly: mm10; hg19
Supplementary files format and content: Genome coverage tracks were generated using the pileup function from MACS2.
Supplementary files format and content: bigWig files showing the genome coverage for merged spike-in normalised biological replicates were generated using the pileup function from MACS2.
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| Submission date |
Jul 09, 2024 |
| Last update date |
Nov 26, 2024 |
| Contact name |
Emilia Dimitrova |
| E-mail(s) |
[email protected]
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| Organization name |
University of Oxford
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| Department |
Biochemistry
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| Lab |
Rob Klose
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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 |
GPL19415 |
| Series (2) |
| GSE271891 |
The PNUTS phosphatase complex controls transcription pause release [ChIP-Seq] |
| GSE271892 |
The PNUTS phosphatase complex controls transcription pause release |
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| Relations |
| BioSample |
SAMN42390129 |
| SRA |
SRX25260969 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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