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| Status |
Public on Jun 27, 2018 |
| Title |
SFPQ PARCLIP U2OS |
| Sample type |
SRA |
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| Source name |
U2OS
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Immortalised osteosarcoma cells
passages: 16-20
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| Treatment protocol |
Cells were cultured in normal growth media. Prior to UV crosslinking, cells were supplemented with 4SU for 14 hours. For UV crosslinking, cells were washed with cold PBS and then irradiated with 365 nm UV at 0.15 J/cm2 in a Stratalinker 2400.
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| Growth protocol |
Both HeLa and U2OS cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with high glucose (4500mg/L), L-glutamine (584mg/L) and sodium pyruvate (110mg/L)(Life Technologies, cat# 11995065), and supplemented with 10% Fetal Calf Serum (Serana, cat# FBS-AU-015) and 100U/ml Penicillin/Streptomycin (Gibco, cat# 15140122). Cells were grown at 37°C in 5% CO2 in a humidified incubator.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Following UV crosslinking, cells were harvested, lysed in NP-40 lysis buffer and passed through a 32gauge needle. Lysates were cleared by centrifugation and then incubated with RNase T1 (Fermentas). Lysates were pre-cleared using Protein G Dynabeads (Invitrogen), after which RNA-protein complexes containing NONO were immunoprecipitated with NONO antibody (Monoclonal Antibody (MAb) Facility) conjugated to Protein G Dynabeads (Invitrogen). Following immunoprecipitation, beads were washed and then resuspended in dephosphorylation buffer and Calf intestinal alkaline phosphatase (New England Biolabs). Beads were then washed and incubated in polynucleotide kinase buffer and RNA was radiolabelled with Y-32P-ATP (Perkin Elmer). Radiolabelled protein-RNA complexes were removed from Dynabeads then resolved by SDS-PAGE on a NuPAGE Novex 4-12% Bis-Tris Protein Gel. Using 32P radiography RNA-protein complexes corresponding to the sizes of NONO and SFPQ were excised from the gel, and electroeluted in D-Tube Dialyzer midi tubes (Merck Millipore). Electroeluant was digested with Proteinase K (Fermentas) and RNA was then extracted from samples using miRNAeasy Micro Kit (Qiagen).
RNA extracted from PAR-CLIP was converted into a cDNA library using TruSeq small RNA Kit v2 (Illumina) with a size selection range 145-327 bp (RNA of 18-200 nt) and Illumina sequencing with 50bp single reads.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Cutadapt 1.8.1 was used to remove the sequencing adapters and for quality control (--max-n 0 -m 20 -q 20,20).
Sequenced reads were aligned to the human reference genome (hg38) using bowtie 1.1.1 allowing up to one mismatch, and uniquely mapped reads were retained (-v 1 -m 1).
In the uniquely mapped reads, identical reads (PCR duplicates) were collapsed.
Peaks were called using PARalyzer v1.5 with default and recommended parameters as described in the manual.
Genome_build: hg38
Supplementary_files_format_and_content: Bed files were obtained from PARalyzer (OUTPUT_CLUSTERS_FILE) including chromosome, start coordinate , end coordinate, peak ID, PARalyzer score (ModeScore), strand, T->C conversion count, read count
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| Submission date |
Apr 18, 2018 |
| Last update date |
Jun 27, 2018 |
| Contact name |
Archa Fox |
| E-mail(s) |
archa.fox@uwa.edu.au
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| Organization name |
University of Western Australia
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| Street address |
35 Stirling Highway
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| City |
Nedlands |
| ZIP/Postal code |
6009 |
| Country |
Australia |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE113349 |
PARCLIP analysis of NONO and SFPQ from U2OS and Hela cells |
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| Relations |
| BioSample |
SAMN08950915 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3104133_SFPQ_2_U2OS_C6GR9ANXX_GTCCGC_L007_HRR0000007_R1.fastq.noadapt_m20_q2020.hg38_bowtie_v1_m1.filtered.sam.collapsed.PARalyzer_result.bed.all.bed.gz |
7.1 Mb |
(ftp)(http) |
BED |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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