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Sample GSM4110760

Query DataSets for GSM4110760

Status

Public on Jan 20, 2020

Title

RPF_control_rep3

Sample type

SRA

Source name

hTERT-RPE-1

Organism

Homo sapiens

Characteristics

cell type: hTERT-immortalized retinal pigment epithelial cell line
treatment: control-siRNA (100 nM)
passages: 12-14

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)

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).

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)

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina HiScanSQ

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

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

Submission date

Oct 07, 2019

Last update date

Jan 20, 2020

Contact name

Astrid Haas

Organization name

WWU Münster

Department

Biochemistry

Street address

Wilhelm-Klemm-Str.2