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| Status |
Public on May 19, 2015 |
| Title |
IP RNAPII (Int11-KD) |
| Sample type |
SRA |
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| Source name |
HeLa-LTR-Luciferase cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: HeLa-LTR-Luciferase cells
genotype/variation: depleted of Int11
chip antibody: Anti-RNAPII total
chip antibody vendor: Santa Cruz
chip antibody cat.#: sc-899
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| Treatment protocol |
Chromatin was purified from 100 millions HeLa-LTR-Luciferase cells for each sample
Chromatin immunoprecipitation (ChIP) was performed using 10 µg antibody resulting in the following amounts of DNA pellets:
Anti-RNAPII total (Santa Cruz) Replicate A: 53.7ng. Replicate B: 71.1ng.
Anti-H3K4me3 (Abcam): Replicate A: 1290ng. Replicate B: 1251ng.
Anti-NELF-E (Millipore): Replicate A: 21ng. Replicate B: 27ng.
Anti-Spt5 (BD Transduction Laboratories): Replicate A: 18ng. Replicate B: 24ng.
Anti-INTS3 (Bethyl Laboratories): Replicate A: 81ng. Replicate B: 81ng.
Anti-INTS11:(Bethyl Laboratories) Replicate A: 10.8ng. Replicate B: 13.2ng.
Anti-INTS13 (Bethyl Laboratories): Replicate A: 33ng. Replicate B: 43.5ng.
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| Growth protocol |
Exponentially growing HeLa-LTR-Luciferase cells
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin immuno-precipitations (ChIPs) were performed as described in Whyte et al. 201228 using ~100 x 106 HeLa-LTR-Luciferase cells as starting material with the exception of an additional nuclei purification step. Briefly, cells were thawed and resuspended in 1ml Buffer A (0.3M SUCROSE, 60mM KCl, 15mM NaCl, 5mM MgCl, 0.1 mM EGTA, Tris-HCl pH=7.5, 0.2mM PMSF), 1ml Buffer B (0.3M SUCROSE, 0.2% NP40, 60mM KCl, 15mM NaCl, 5mM MgCl, 0.1 mM EGTA, Tris-HCl pH=7.5, 0.2mM PMSF) was then added and incubated for 7min on ice, laid over a 8ml cushion of Buffer C (1.2M SUCROSE, 0.2% NP40, 60mM KCl, 15mM NaCl, 5mM MgCl, 0.1 mM EGTA, Tris-HCl pH=7.5, 0.2mM PMSF) and spinned for 20min at 3000rpm on 4 C. Pelleted nuclei were resuspended in 3ml Lysis buffer, incubated for 1 hour, sonicated (Misonix sonicator with the following settings: micro tip, 30sec on, 2min off, amplitude 70, 7min total sonication time). 10ng, as quantified by Qubit dsDNA HS Assay Kit (Life Technologies), of input and of immuno-precipitated-material was used for library preparation.
Librairies were prepared using Illumina ChIP-Seq sample prep kit (non-multiplexed libraries) or TruSeq ChIP Sample Prep Kit (multiplexed libraries) according to manufacturer’s instructions. Image analyses and base calling were performed using the HiSeq Control Software and Real-Time Analysis component. Data quality was assessed using fastqc from the Babraham Institute and the Illumina software SAV (Sequence Analysis Viewer). De-multiplexing and alignment were performed using Illumina's sequencing analysis software (CASAVA 1.8.2).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Sample 15
processed data file: Profile_RNAPII_Int11_KD_TES.tar.gz, Profile_RNAPII_Int11_KD_TSS.tar.gz
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| Data processing |
Basecalls performed using CASAVA version 1.8.2
Burrows Wheeler Alignment tool (BWA) software (default parameters) was used for base-calling
ChIP peaks were analyzed using MACS (Feng et al., 2012) or MACS2 with respect to input control (MACS14* processed data files).
For RNAPII, rMAT package was utilized by counting normalized (RPM) ChIP-Seq reads in the indicated windows with respect to transcription start sites (TSS; -200 to +2000) or to termination sites (TES; -2000 to + 200) using bioconductor transcript database of UCSC (BSgenome.Hsapiens.UCSC.hg19; Profile_RNAPII*.tar.gz)
Processed Files were normalized to the corresponding input as followed: H3K4me3 (MACS14_BN_H3K4me3_vs_Input.tar.gz= sample 2 normalized to sample 1 ); Int3: (MACS14_7A_Int3_vs_Input.tar.gz= sample 4 normalized to sample 3) ; Asun/Int13 (MACS14_7A_Asun_vs_Input.tar.gz= sample 7 normalized to sample 3); NELF: (MACS14_7A_NELF_vs_Input.tar.gz= sample 5 normalized to sample 3); RNAPII: (MACS14_BN2_PolII_vs_Input.tar.gz= sample 9 normalized to sample 8); SPT5: (MACS14_7A_Spt5_vs_Input.tar.gz= sample 6 normalized to sample 3); Int11: (MACS14_X_Int11_vs_Input.tar.gz= sample11 normalized to sample 10)
Genome_build: Hg19
Supplementary_files_format_and_content: Processed data files are directly from the MACS output, with .bed and .xls files.
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| Submission date |
Aug 21, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Olivier Cuvier |
| E-mail(s) |
[email protected]
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| Organization name |
CNRS
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| Department |
LBME
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| Lab |
Chromatin Dynamics & Cell proliferation
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| Street address |
118 route de Narbonne
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| City |
Toulouse |
| ZIP/Postal code |
31062 |
| Country |
France |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE60584 |
Integrator complex regulates NELF-mediated RNA Polymerase II pause/release and processivity at coding genes [ChIP-seq] |
| GSE60586 |
Integrator complex regulates NELF-mediated RNA Polymerase II pause/release and processivity at coding genes. |
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| Relations |
| BioSample |
SAMN03000058 |
| SRA |
SRX684301 |
| 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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