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| Status |
Public on Dec 26, 2018 |
| Title |
siLuc (rep 2) |
| Sample type |
SRA |
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| Source name |
cervix
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| Organism |
Homo sapiens |
| Characteristics |
cell type: HeLa
cell type: epithelial
sirna treatment: 48h siLuciferase (Sigma-Aldrich)
chemical treatment: Double thymidine block
cell cycle stage: Early S-phase
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| Treatment protocol |
About 50 % confluent HeLa cells were transfected with a SMARTpool of siRNA for HSE or CPSF73 (GE Dharmacon), or with a negative control siRNA (Luciferase), using the Lipofectamine™ RNAiMAX transfection reagent (Invitrogen) following the manufacturer’s instructions. Cells were transfected with a final concentration of 10 nM siRNA and knockdown efficiency was assessed 48 hours post transfection by western blot analysis. Transfected HeLa cells were synchronized using the double thymidine block method
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| Growth protocol |
HeLa cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, and 2 mM L-glutamine, at 37°C in a humidified incubator (5% CO2).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Approximately 3x106 cells were resuspended in 500 µL of ice-cold RLB buffer, (10 mM Tris-Hcl, pH 7.5, 140 mM NaCl, 0.5% NP40, 1.5 mM MgCl2), and lysed by adding an equal volume of RLB buffer with 24% (m/v) sucrose. Nuclei were collected by centrifugation (14,000 x g) at 4°C for 10 min. Isolated nuclei were resuspended in 120 µl of NUN1 buffer, (20 mM Tri-HCl, pH 7.9, 75mM NaCl, 0.5 mM EDTA, 50% Glycerol, 0.125 mM PMSF, 1mM DTT), followed by addition of 1.2 ml NUN2 buffer, (20 mM HEPES-KOH pH 7.6, 7.5mM MgCl2, 0.2 mM EDTA, 300 mM NaCl, 1 M urea, 1 % NP40, 1 mM DTT). Nuclei were incubated for 15 minute on ice with mixing by vortexing for 5 seconds every 5 min. Chromatin pellets were precipitated by centrifugation (14,000 x g) at 4°C for 10 min and then resuspended in 200 ml HSB buffer, (10 mM Tris-HCl, pH 7.5, 500 mM NaCl, 10 mM MgCl2), in presence of 4 U TURBO DNase (Ambion) and incubated at 37°C for 20 minutes. Followed a 20 minutes proteinase K treatment (Roche) at 37°C, chromatin RNA (chrRNA) was phenol-chloroform extracted and ethanol precipitated. RNA was resuspended in 200 µL of 1x TURBO DNAse buffer in presence of 4 U TURBO DNase and incubated at 37°C for 30 minutes. The entire procedure of extraction, precipitation and DNase treatment was repeated twice. RNA was then extracted and precipitated again, prior to solubilisation in 75 µL of RNase free water. Prior to RNA libraries preparation, rRNAs were depleted using Ribo-Zero Magnetic kit (Illumina) from 2.5 µg of ChrRNA.
Libraries were prepared starting from chromatin RNA using the NEBNext Ultra Directional RNA Library Prep kit for Illumina (NEB) following the manufacturer’s instructions
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
Chromatin RNA
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| Data processing |
RNA-sequencing reads were trimmed using Cutadapt and then mapped to the human reference genome with Tophat 2.0.13.
Aligned reads were processed to only include properly paired, properly mapped reads with no more than 2 mismatches using SAMtools.
Data were scaled to library size (genomeCoverageBed) using Bedtools.
Genome_build: hg38
Supplementary_files_format_and_content: BigWig file containing read densities from RNA-seq experiments.
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| Submission date |
Feb 08, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Nicholas Proudfoot |
| E-mail(s) |
nicholas.proudfoot@path.ox.ac.uk
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| Organization name |
Sir William Dunn School of Pathology, University of Oxford
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| Street address |
South Parks Road
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| City |
Oxford |
| ZIP/Postal code |
OX1 3RE |
| Country |
United Kingdom |
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| Platform ID |
GPL20301 |
| Series (1) |
| GSE94686 |
Biosynthesis of histone messenger RNA employs a specific 3' end endonuclease |
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| Relations |
| BioSample |
SAMN06312339 |
| SRA |
SRX2545560 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2480871_Rep2_siLUC_neg.bw |
155.9 Mb |
(ftp)(http) |
BW |
| GSM2480871_Rep2_siLUC_pos.bw |
162.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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