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| Status |
Public on Aug 20, 2015 |
| Title |
hDIS3_PAR-CLIP |
| Sample type |
SRA |
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| Source name |
HEK293 Flip-In T-Rex cell line
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| Organism |
Homo sapiens |
| Characteristics |
variant: DIS3D487N
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| Treatment protocol |
Expression of exogenous genes was induced by addition of doxycycline at a final concentration of 100ng/ml. Simultaneously, 4-thiouridine (4-sU) was added to the culture medium at a final concentration of 100µM. The 4-sU molecules incorporate into nascent RNA allowing for subsequent photoreactive nucleoside-labelled RNA-protein cross-linking. After 24 hours cells were washed with ice-cold PBS, irradiated with 0.12J/cm2 of 365nm UV light (CL-1000 UV Crosslinker; UVP).
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| Growth protocol |
The stable inducible HEK293 Flp-In T-Rex cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco) and penicillin/streptomycin (Sigma Aldrich) at 37°C in a 5% CO2. Cells were cultured in a Petri dish (145 x 20mm) to approximately 80% confluency.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were scraped off and centrifuged at 400 x g for 5 min. at 4°C. 5 plates yielded 1 ml of wet cell pellet, which was sufficient for one experiment. Cell pellets were resuspended in lysis buffer, sonicated and centrifuged at 16000 x g for 15 min at 4°C. To obtain RNA of an appropriate size, cleared extracts were treated with 5U/ml of RNAse T1 (Ambion) for 10 min at 22°C with shaking at 150 rpm and cooled on ice for 5 min. Cell lysates containing RNA protein complexes were then mixed with urea at a final concentration of 0.5M and added to 50µl of freshly washed magnetic beads coated with GFP-trap antibodies and were incubated for 1 hour at 4°C with rotation. Co-immunoprecypitation was followed by 3 washes with high salt wash buffer (HS buffer), one wash with low salt wash buffer (LS buffer) and 1 wash with PNK buffer. Beads were resuspended in 50µl of PNK buffer and treated with 35U alkaline phosphatase (NEB) in the presence of RNAse inhibitor (Ribolock; Thermo Scientific) for 20 min at 37°C with shaking at 1100 rpm in Thermomixer (Eppendorf) and immediately washed twice with PNK buffer and twice with ligation buffer. The resulting short RNA fragments, dephosphorylated at their 3’ ends and still crosslinked to proteins, were ligated with a pre-adenylated linker Ra3 (Illumina). The reaction was done by treating with 400U of T4 RNA Ligase 2, truncated (NEB) overnight at 16°C with shaking at 1100 rpm in 50µl of linker ligation buffer in the presence of 20% PEG 400 (Sigma) and 1mM DTT (Sigma). This step was followed by washing once with HS buffer, twice with LS buffer and once with PNK buffer. RNA molecules cross-linked to immunoprecipitated proteins were then radiolabeled at their 5’ ends. Beads were resuspended in 50µl of PNK buffer with 15U of T4 polynucleotide kinase (NEB) and 5µCi [32P]-γ-ATP and incubated at 37°C with shaking at 1100 rpm for 15 min. Then cold ATP was added and the incubation continued for 15 more minutes. Beads with radiolabeled RNA were then washed 4 times with PNK buffer, resuspended in NuPAGE LDS loading buffer (Life Technologies) and incubated for 10 min at 70°C with shaking 1100 rpm to denaturate and release RNAs-RBPs complexes. Then DTT was added to the final concentration of 65mM. RNAs-protein complexes were resolved by size using 4-12% NuPAGE Bis-Tris gel (Life Technologies) in MOPS SDS running buffer (Invitrogen) and transferred from the gel onto Protran Nitrocellulose membrane (Whatman) by wet-blotting (30V, 1h) in a Invitrogen XCell II Blot Module using NuPAGE transfer buffer (Invitrogen) according to the manufacturer's instructions. Membranes were stained with Ponceau S Red (Sigma-Aldrich; 0.1% in 3% acetic acid) and exposed to a phosphoscreen for 30 min. The RNA-protein complexes migrated above the expected MW of the protein, and appeared as a smeary signal. The area of the membrane in which radioactivity appeared was cut into small slices and placed in 400µl of PK buffer. RNAs were released by proteinase K digestion: Proteinase K (BioLine) was added to the samples at a final concentration of 500µg/ml and was incubated at 37°C at 1100 rpm for 1h. The solution containing RNAs was then mixed with phenol:chloroform and shaken at 1100 rpm. Phases were separated by spinning for 5 min at 16000 x g at room temperature. The aqueous layer was mixed with chloroform (1:1), shaken and centrifuged as described above. RNAs were precipitated overnight in the presence of Glycoblue (Ambion), NaAc, pH=5.2, and ethanol. Finally, RNA molecules were purified using magnetic beads (Agencourt AMPure XP; Beckman Coulter) according to the manufacturer's protocol.
RNA libraries were prepared from the obtained RNA using ScriptSeq™ v2 RNA-Seq Library Preparation Kit (Epicentre) according to the manufacturer's instructions. A cDNA synthesis primer, reverse PCR primer containing user-defined barcode and pre-adenylated linker Ra3 were from TruSeq Small RNA Sample Preparation Kit (Illumina).
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 1500 |
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| Description |
library strategy: PAR-CLIP
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| Data processing |
Basecalls and demultiplexing were performed using CASAVA version 1.8.2
Adapters and reads shorter than 12 nt. and with quality lower than 20 were removed by cutadapt-1.2.1
Reads were mapped using RUMv2.05_05. The program requires left and right reads to be the same length, so reads has been striped adequately. The information about known transcripts was supplied to the program. It contained i) Gencode v18 transcript data ii) supplemented to Gencode v18 data tRNA coordinates from tRNA scan program iii) previously published PROMPT data which is a set 3kB upstream regions from the selected 2471 genes iv) snoRNA precursors defined by us for intronic snoRNA as regions from the end of the snoRNA to the beginning of the exon.
Only uniquely mapped reads have been counted.
New transcripts were assembled from deeply sequenced libraries from PIN RNB double mutant using Cufflinks v 2.1.1. Reads from three replicates were assembled separately. We applied all default parameters with exception of '--max-bundle-length' increased to 10^7 and '--max-bundle-frags' increased to 10^7. We used cuffmerge from cufflinks package to merge assemblies from the replicates.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include raw counts of sequencing reads for each sample replicate; bedgraph files were generated using RUMv2.05_05; gtf file was generated using cuffmerge from Cufflinks v 2.1.1.
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| Submission date |
Dec 18, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Andrzej Dziembowski |
| E-mail(s) |
andrzejd@ibb.waw.pl
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| Phone |
+48 22 5922033
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| Organization name |
Institute of Biochemistry and Biophysics Polish Academy of Sciences
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| Lab |
Laboratory of RNA Biology and Functional Genomics
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| Street address |
Pawinskiego 5A
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| City |
Warszawa |
| ZIP/Postal code |
02-106 |
| Country |
Poland |
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| Platform ID |
GPL18460 |
| Series (1) |
| GSE64332 |
Human DIS3 shapes the RNA polymerase II transcriptome degrading variety of unwanted transcripts. |
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| Relations |
| BioSample |
SAMN03268751 |
| SRA |
SRX817375 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1568713_PARCLIP_1_minus.bedgraph.gz |
5.7 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM1568713_PARCLIP_1_plus.bedgraph.gz |
5.8 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM1568713_PARCLIP_2_minus.bedgraph.gz |
3.5 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM1568713_PARCLIP_2_plus.bedgraph.gz |
3.5 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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