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| Status |
Public on Aug 15, 2014 |
| Title |
pUPF1_2 |
| Sample type |
SRA |
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| Source name |
Human embryonic kidney (HEK) 293T cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T
cell type: Human embryonic kidney
ip antibody: anti-p-UPF1(S1116) (Millipore, anti-phospho-Upf1(Ser1127))
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| Growth protocol |
HEK293T cells (24 x 107/3 x 150-mm dishes) were incubated in 200 nM okadaic acid for three hours.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cellular RNAs bound by p-UPF1 were immunoprecipitated using anti-p-UPF1(S1116) (Millipore, anti-phospho-Upf1(Ser1127)) and Dynabeads protein A magnetic beads (Life Technologies). The RNA in bead-bound RNA p-UPF1(S1116) complexes was digested to primarily 100 nts by incubation for 30 min at 4°C with RNase I (1U/l; Life Technologies); for RNA size estimations after RNase I digestion, ~200 ng of RNA were radio-labeled using 32P-ATP (Perkin Elmer) and T4 polynucleotide kinase (New England Biolabs) and subsequently visualized using a Typhoon 9410 Variable Mode Imager (GE Healthcare) after electrophoresis in 6M urea 15% polyacrylamide. After extensive washing, bound complexes were eluted using IP elution buffer (Kurosaki and Maquat 2013), and RNA fragments were purified using TRIzol Reagent and then separated in 6M urea 15% polyacrylamide in parallel with a DynaMarker Prestain Marker for Small RNA (BioDynamics Laboratory). Small-range RNAs (~25-40 nts) were excised from 6M urea 15% polyacrylamide and agitated overnight at 25°C in RNA extraction buffer (20 mM Tris, 300mM sodium acetate, 2 mM EDTA, 0.2 % v/v SDS). RNAs were eluted from 6M urea 15% polyacrylamide gel using a Coaster Spin-X column (Corning) and further purified using TRIzol Reagent followed by ethanol precipitation. In parallel, control IPs using rabbit IgG were performed. Additional control samples were prepared without IP.
Purified 25-40-nt RNA fragments were treated with recombinant shrimp alkaline phosphatase (New England Biolabs) to remove 3'-phosphates and subsequently phosphorylated at 5'-hydroxyl groups using T4 polynucleotide kinase (New England Biolabs). Phosphorylated RNA fragments were purified with RNeasy Mini Columns (Qiagen). A 3'-adenylated adapter was ligated to the phosphorylated RNA fragments using truncated T4 RNA ligase (New England Biolabs). An RT primer was annealed to the adapted RNAs to prevent adapter self-ligation, followed by 5' RNA adapter ligation using T4 RNA ligase (New England Biolabs). After RT of adapter-ligated RNAs, cDNAs were amplified using 15 PCR cycles. Amplified cDNAs were purified in 8% polyacrylamide, and the quality and quantity of cDNAs were assessed using an Agilent Bioanalyzer and qPCR. cDNAs were then sequenced using the Illumina HiSeq 2500 platform.
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
The 3'-adapter sequence was first removed (using fastx_clipper -a TGGAATT), and reads with a length of <15 nts were discarded.
Reads were mapped to the human genome (hg19) using Bowtie2 (local mode) with parameters "--local -5 4 -3 4". Reads with a mapping quality score (MAPQ) of ≥10 were selected for further analysis.
mRNA abundance was measured using reads per kilobase per million reads (RPKM) based on exonic regions of RefSeq sequences.
Genome_build: hg19
Supplementary_files_format_and_content: uniquely mapped reads, the 5th column in bed file represents read number
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| Submission date |
Aug 04, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Wencheng Li |
| Organization name |
PTC Therapeutics
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| Street address |
100 Corporate Count
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| City |
South Plainfield |
| State/province |
NJ - New Jersey |
| ZIP/Postal code |
07080 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE60045 |
A post-translational regulatory switch on UPF1 controls targeted mRNA degradation. |
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| Relations |
| BioSample |
SAMN02950997 |
| SRA |
SRX668821 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1464150_pUPF1_2.bed.gz |
1.6 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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