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Sample GSM3104133 Query DataSets for GSM3104133
Status Public on Jun 27, 2018
Title SFPQ PARCLIP U2OS
Sample type SRA
 
Source name U2OS
Organism Homo sapiens
Characteristics cell type: Immortalised osteosarcoma cells
passages: 16-20
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.
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.
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.
 
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina HiSeq 2000
 
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
 
Submission date Apr 18, 2018
Last update date Jun 27, 2018
Contact name Archa Fox
E-mail(s) archa.fox@uwa.edu.au
Organization name University of Western Australia
Street address 35 Stirling Highway
City Nedlands
ZIP/Postal code 6009
Country Australia
 
Platform ID GPL11154
Series (1)
GSE113349 PARCLIP analysis of NONO and SFPQ from U2OS and Hela cells
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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