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Status |
Public on Jan 20, 2020 |
Title |
RPF_PDCD4-KD_rep3 |
Sample type |
SRA |
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Source name |
hTERT-RPE-1
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Organism |
Homo sapiens |
Characteristics |
cell type: hTERT-immortalized retinal pigment epithelial cell line treatment: PDCD4-siRNA2 (100 nM) passages: 12-14
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Treatment protocol |
hTERT-RPE-1 cells were reversely transfected with siRNA via Lipofectamine RNAiMAX reagent and cultured for 24 h (PDCD4-siRNA: GCUUCUUUCUGACCUUUGU; control-siRNA (targeting renilla luciferase): AAACAUGCAGAAAAUGCUG)
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Growth protocol |
hTERT-RPE-1 adherent cells were cultured at coated plastic (T75 flask, Greiner Bio-One or cell culture plates, 10 cm, Corning (Falcon)) in DMEM/Ham's F12 medium (Biochrom).
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Extracted molecule |
polyA RNA |
Extraction protocol |
For ribosome proliferation RPE cells were incubated for 24 h after transfection with siRNA (Pdcd4 siRNA-2 or control siRNA (100 nM), reverse transfection with Lipofectamine RNAiMAX) and media was changed two hours before harvesting. To stabilize elongating ribosomes, cells were treated with 100 µg/mL cycloheximide (CHX) for 5 min at 37 °C, following a washing step with ice cold PBS (+ 100 µg/mL CHX). Cells were lysed in lysis buffer (10 mM Tris-HCl (pH=7.4), 10 mM MgCl2, 100 mM NaCl, 1% Triton, 1 mM DTT, 100 µg/mL CHX) per sample (sample = 8 dishes á 10 cm), and cell debris pelleted at 4 °C, 10 000 g for 3 min. 10 OD260 units of extract were treated with 900 U RNase I (Ambion)(+ 0.5 % deoxycholate) for 20 min at 22 °C, 800 rpm in a Thermomixer. The reaction was stopped by addition of 240 U SUPERase In (Ambion)(+ 0.5 % deoxycholate) and extracts were resolved by centrifugation at 4 °C, 35 000 rpm for 3 h in a SW-41 Ti swinging-bucket rotor (Beckman Coulter) on 10-50% sucrose density gradients (20 mM Tris-HCl (pH=7.5), 10 mM MgCl2, 100 mM NH4Cl, 2 mM DTT, 100 µg/mL CHX). Gradients were fractionated at 0.75 mL/min with continuous monitoring of OD254 values using a Biocomp Instruments Gradient Station (Teledyne Isco.). Monosome fractions were collected and, following addition of SDS 1%, flash-frozen and stored at -80 °C. RNA was isolated from gradient fractions by the hot acid phenol method (1-bromo-3-chloropropane used instead of chloroform), and ribosome footprints were purified from monosome RNA by size selection of 28-30 nt fragments (excluding major band around 31 nt) on 15 % polyacrylamide, 8 M urea, 1xTBE gels. rRNA depletion with RiboZero Kit was skipped, since contaminating reads were removed by aligning against a reference containing all the rRNA of the organism of interest RNA libraries for ribosome profiling were prepared accoding to the protocol from Lecanda2016 (Lecanda A, Nilges BS, Sharma P, Nedialkova DD, Schwarz J, Vaquerizas JM, Leidel SA (2016). Dual randomization of oligonucleotides to reduce the bias in ribosomeprofiling libraries. Methods 107:89–97)
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Library strategy |
OTHER |
Library source |
transcriptomic |
Library selection |
other |
Instrument model |
Illumina HiScanSQ |
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Description |
ribosome protected fragments (RPF) siRNA_RPE_RPF_res.txt siRNA_RPE_RPF_resLA.txt siRNA_RPE_RPF_normalized.txt siRNA_RPE_RPF_mRNA_res.txt
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Data processing |
Library strategy: Ribosome profiling Reads were demultiplexed with Illumina's bcl2fastq for ribosome profiling: adapter sequences were clipped with the FASTX-Toolkit (Hannon Lab, version 0.0.13.) for ribosome profiling: 4 randomized nucleotides were trimmer using the FASTX-Toolkit (Hannon Lab, version 0.0.13.) for ribosome profiling: rRNA was depleted by mapping against human rRNA using bowtie version 1.0.0 mapping to UCSC cannonical ORFs was performed with bowtie version 1.0.0 count tables were created using a custom script and differential expression determined with DESeq2 Genome_build: GRCh38 Supplementary_files_format_and_content: tab-delimited txt files including standard DESeq2 output (baseMean, log2FoldChange (lfc), lfcSE, statx, pvalue, padj) *normalized.txt: normalized reads *resLA.txt: fully analyzed data; tested for significantly unchanged genes *res.txt: fully analyzed data; tested for significantly changed genes siRNA_RPE_RPF_mRNA_res.txt: combined file for RNA-seq and ribosome profiling fully analyzed data; tested for significantly changed genes
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Submission date |
Oct 07, 2019 |
Last update date |
Jan 20, 2020 |
Contact name |
Astrid Haas |
Organization name |
WWU Münster
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Department |
Biochemistry
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Street address |
Wilhelm-Klemm-Str.2
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City |
Münster |
ZIP/Postal code |
48149 |
Country |
Germany |
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Platform ID |
GPL15456 |
Series (1) |
GSE138533 |
Analysis of PDCD4 knockdown effects by high throughput sequencing of a human epithelial cell line |
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Relations |
BioSample |
SAMN12985344 |
SRA |
SRX6959645 |
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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