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| Status |
Public on May 12, 2016 |
| Title |
PAR-CLIP_GSC_r1 |
| Sample type |
SRA |
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| Source name |
Glioma stem cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MGG8
tumor state: Cancer stem cells
growth: Neurobasal medium (Invitrogen) supplemented with L-glutamine (Glutamax, Gibco), B27 supplement (Invitrogen), N2 supplement (Invitrogen), 20 ng/mL recombinant human EGF (R&D Systems), 20 ng/ml recombinant human FGF2 (R&D Systems).
treatment: Cells were grown overnight in the presence of 100 uM 4-SU
sample type: IMP2 bound RNA
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| Extracted molecule |
total RNA |
| Extraction protocol |
100e6 GSC with overnight 4SU incubation were dissociated mechanically by pipetting vigorously, pelleted at 500 rcf for 5 minutes and washed once in cold PBS. After repeating centrifugation supernatant was removed, cells were resuspended in cold PBS, spread out in a thin liquid sheet on 145mm plates and crosslinked at 365nm and a total energy of 0.36 J/cm2. After centrifugation, pellet was resuspended in lysis buffer (50 mM HEPES pH 7.5, 150 mM KCl, 5 mM CaCl2, 1 % NP-40) and incubated 15 minutes on ice. Lysate was treated with 20 U/mL Fermentas RNase T1 and 15 U/mL Fermentase RNase free DNase I for 15 minutes at RT and then sonicated for 15 cycles (30 sec on / off) on a Diagenode Bioruptor on high. Lysate was cleared at 21’000 rcf and 4 °C for 10 minutes. IP was performed for 3 hours with 20 ug rabbit pAb MBL Anti-IGF2BP2 (RN008P) preincubated for 1 hour at RT with 100 uL Invitrogen protein A dynabeads. Beads were washed 3 times in lysis buffer and treated with RNase T in 100 uL at 20 U/uL for 15 minutes at RT. After three more lysis buffer and two dephosphorylation buffer washes (50 mM Tris-HCl pH 7.9, 100 mM NaCl, 10 mM MgCl2) RNA was dephosphorylated at 37 °C in 100 uL NEB buffer 3 with 0.5 U / uL CIP for 10 minutes. 500 uL lysis buffer were added for beads to rebind during 10 minutes on ice. Three more lysis buffer washes and two PNK-buffer (70 mM Tris-HCl, pH 7.6, 10 mM MgCl2) washes were followed by phosphorylation with radioactive 32P at 37 °C in 100 uL of PNK buffer with 5 mM DTT, 17 nM (10 uCi) 32P-ATP and 0.5 U/uL PNK. After 30 minutes 5 uM normal ATP were added for additional 5 minutes. 1 mL of lysis buffer was added and beads are allowed to rebind during 10 minutes on ice. Beads were washed 5 times with lysis buffer, boiled in SDS-DTT solution and run on a NuPage Novex 4-12 % bis-tris gel for 1.5 hours at 150 V in MOPS buffer. Gel was imaged for 30 minutes with a phosphorimaging cassette and bands of interest excised. Elution of Protein-RNA complex was performed three times in 400 uL MOPS buffer using d-tube dialyzer midi MWCO 3.5 kDa each for 1 hours at 100 V and 5 minutes at 100 V reversed polarity. All subsequent steps were carried out exactly as described Hafner M. et al 2010.
Library was prepared similary to Illumina small RNA protocol. We adhered to Hafner et al 2010. Library was amplified for 18 cycles during PCR.
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
PARCLIP_IMP2s_r1.bed
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| Data processing |
Adaptor trimming, custom python script
Genomic alignment with bowtie 0.12.9 --sam -n 1 -l 20 -m 100 --best
Sam to bam conversion , samtools 0.1.9 samtools view -b -S
Sort and index, samtools 0.1.9 samtools sort, samtools index
Binding cluster calling, wavClusteR 2.2.0, R 3.2.0, prob. given by bayes, min. coverage 10 (5) for rep. 1 (2), min. cluster 10
Genome_build: hg19
Supplementary_files_format_and_content: Stranded BED-File of detected binding clusters, scores are log2 cluster coverage scaled to 0-1000
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| Submission date |
Feb 01, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Tommy Beat Schlumpf |
| Organization name |
ETH Zürich
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| Department |
D-BSSE
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| Lab |
Paro
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| Street address |
Mattenstrasse 26
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| City |
Basel |
| State/province |
Basel-Stadt |
| ZIP/Postal code |
4058 |
| Country |
Switzerland |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE73845 |
Functional characterization of RNA-binding protein IMP2 in primary Glioma cell lines [HTS] |
| GSE73847 |
Functional characterization of RNA-binding protein IMP2 in primary Glioma cell lines |
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| Relations |
| BioSample |
SAMN04450717 |
| SRA |
SRX1556055 |
| 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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