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| Status |
Public on Aug 04, 2022 |
| Title |
IP RNAPII Ctrl R2 |
| Sample type |
SRA |
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| Source name |
Neuroblastoma SH-SY5Y cell line (ECACC)
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| Organism |
Homo sapiens |
| Characteristics |
cell line: SH-SY5Y
transfected sirna: Negative control siRNA anti-luciferase
extract: RNA Pol II IP (F12 antibody)
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| Treatment protocol |
Cells were transfected for 48h with siRNAs at a final concentration of 20 nM with lipofectamine RNAimax (ThermoFischer Scientific).
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| Growth protocol |
Cells were cultured in 4,5 g/L Glucose DMEM/F-12 (ThermoFischer Scientific) supplemented with 10% (v/v) fetal bovine serum (ThermoFischer Scientific), and 2 mM L-Glutamine (ThermoFischer Scientific) in 5% CO2 at 37°C.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For each of the 3 replicates of calibrated RNAPII ChIP-seq, we pooled 4 independent IP, each of them performed on 40 µg of chromatin supplemented with 32 ng of Drosophila spike-in chromatin (Active Motif #53083), with 4 µg of mouse anti-Pol II F-12 antibody (F-12, sc55492, Santa Cruz) and 2 µg of spike-in antibody (Active Motif #61686).
Libraries and sequencing was performed by the GenomEast platform (Illkirch, France). ChIP samples were purified using Agencourt AMPure XP beads (Beckman Coulter) and quantified with the Qubit (Invitrogen). ChIP-seq libraries were prepared from 10 ng of double-stranded purified DNA using the MicroPlex Library Preparation kit v2 (C05010014, Diagenode, Seraing, Belgium), according to manufacturer's instructions.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
Replicate 2
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| Data processing |
High throughput sequencing of 50 bp single-end reads was carried out on an Illumina HiSeq 4000 platform.
Image analysis and base calling were performed using RTA 2.7.3 and bcl2fastq 2.17.1.14.
The approach is similar to a standard ChIP-seq analysis, except that the presence of a defined amount of exogenous chromatin (from Drosophila) in each sample allows a normalization of the experimental samples and thus a more accurate comparison of RNAPII occupancy across samples. After trimming and removing adaptors, reads were then competitively mapped on a concatenation of the two genomes (the reference human genome hg19, and the calibration drosophila genome dm6) and assigned to their corresponding genome. Calibration of the read coverage was then performed for each nucleotide position.
Details of the Nextflow pipeline are available in the following GitHub repository: https://gitbio.ens-lyon.fr/LBMC/Bernard/quantitative-nucleosome-analysis/ (tag qChIPseq_polII_cbourgeois https://gitbio.ens-lyon.fr/LBMC/Bernard/quantitative-nucleosome-analysis/-/tags/qChIPseq_polII_cbourgeois and instructions in README_quantitativ-chip.md
Genome_build: hg19
Supplementary_files_format_and_content: BigWigs files
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| Submission date |
Sep 06, 2021 |
| Last update date |
Aug 04, 2022 |
| Contact name |
Hélène Polvèche |
| E-mail(s) |
hpolveche@istem.fr
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| Organization name |
I-Stem
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| Street address |
28, Rue Henri Desbruères
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| City |
Corbeil-Essonnes |
| ZIP/Postal code |
91100 |
| Country |
France |
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| Platform ID |
GPL20301 |
| Series (1) |
| GSE183517 |
Analysis of the genome-wide distribution of RNA Pol II in a neuroblastoma cell line under silencing of RNA helicases DDX5 and DDX17 |
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| Relations |
| BioSample |
SAMN21243969 |
| SRA |
SRX12024640 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5558692_IP_Ctrl_2_fastaCalib_occupancy_norm.bw |
15.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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