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| Status |
Public on Sep 21, 2022 |
| Title |
Input 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 |
transfected sirna: Negative control siRNA anti-luciferase
extract: Chromatin extract (input)
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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 |
Cells were cross-linked (1% formaldehyde), harvested, lysed and sonicated (Covaris S220 ultrasonicator). Chromatin (40 µg per antibody) was diluted and precleared on Protein A/G Dynabeads for 30 min at 4°C under rotation. Beads were removed and 1% of chromatin was taken apart as Input material. Antibodies were added to chromatin and incubated overnight at 4°C under constant mixing, before being bound to Protein A/G Dynabeads for 5h. Immunoprecipitated material was washed once with Low Salt buffer, once with High Salt Buffer, once with LiCl Buffer and twice with Tris/EDTA buffer, before being eluted and reverse crossed-linked overnight at 65°C on Thermomixer (Eppendorf) in the presence of 200 mM NaCl and 20 µg proteinase K (Roche). DNA was then purified by phenol extraction and ethanol precipitation, and quantified for library preparation.
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 s.a., Seraing, Belgium), according to manufacturer's instructions. In the first step, the DNA was repaired and yielded molecules with blunt ends. In the next step, stem-loop adaptors with blocked 5 prime ends were ligated to the 5 prime end of the genomic DNA, leaving a nick at the 3 prime end. The adaptors cannot ligate to each other and do not have single-strand tails, avoiding non-specific background. In the final step, the 3 prime ends of the genomic DNA were extended to complete library synthesis and Illumina compatible indexes were added through a PCR amplification (7 + --- cycles). Amplified libraries were purified and size-selected using Agencourt AMPure XP beads (Beckman Coulter) to remove unincorporated primers and other reagents.
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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 |
Image analysis and base calling were performed using RTA 2.7.3 and bcl2fastq 2.17.1.14.
Sequence reads were mapped to reference genome hg19 using Bowtie 1.0.0 with the following parameters -m 1 --strata --best -y -S -l 40 -p 2.
Genome_build: hg19
Supplementary_files_format_and_content: Wig files were generated using with an in-house script (Variable step, span=25, reads were elongated to 200b).
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| Submission date |
Sep 11, 2020 |
| Last update date |
Sep 21, 2022 |
| Contact name |
Cyril F Bourgeois |
| E-mail(s) |
cyril.bourgeois@inserm.fr
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| Organization name |
ENS de Lyon
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| Department |
LBMC
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| Street address |
46 Allée d'Italie
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| City |
Lyon |
| ZIP/Postal code |
69006 |
| Country |
France |
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| Platform ID |
GPL20301 |
| Series (1) |
| GSE157850 |
Effect of DDX5/DDX17 on genome-wide RNA Polymerase II distribution in SH-SY5Y cells |
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| Relations |
| BioSample |
SAMN16112210 |
| SRA |
SRX9108347 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4776737_wigs_for_CLBS64.wig.gz |
167.9 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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