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Sample GSM2051889 Query DataSets for GSM2051889
Status Public on May 12, 2016
Title PAR-CLIP_diff_GSC_r1
Sample type SRA
Source name Differentiated Glioma stem cells
Organism Homo sapiens
Characteristics cell line: MGG8
tumor state: Differentiated cancer stem cells
growth: Withdrawal of growth factors for 7 days in DMEM with 10% FCS on 100ug/mL poly-D-lysine (Sigma) and 15ug/mL laminin (Sigma) coated plates.
treatment: Cells were grown overnight in the presence of 100 uM 4-SU
sample type: IMP2 bound RNA
Extracted molecule total RNA
Extraction protocol 300e6 differentiated GSC with overnight 4SU incubation were washed once in cold PBS and crosslinked at 365nm and a total energy of 0.15 J/cm2. Cells were scraped and resuspended in cold PBS. After centrifugation at 500 rcf for 5 minutes, 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.
Library strategy RIP-Seq
Library source transcriptomic
Library selection other
Instrument model Illumina HiSeq 2500
Description PARCLIP_IMP2a_r1.bed
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
Submission date Feb 01, 2016
Last update date May 15, 2019
Contact name Tommy Beat Schlumpf
Organization name ETH Zürich
Department D-BSSE
Lab Paro
Street address Mattenstrasse 26
City Basel
State/province Basel-Stadt
ZIP/Postal code 4058
Country Switzerland
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
BioSample SAMN04450718
SRA SRX1556056

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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