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Sample GSM3724995

Query DataSets for GSM3724995

Status

Public on Aug 04, 2022

Title

LogPhase_monosome_rep.2

Sample type

SRA

Source name

ribosome profiling

Organism

Homo sapiens

Characteristics

cell line: HEK293 cells

Treatment protocol

60% confluent cells were subjected to 2h of serum serum starvation (-FBS) or 1h of amino acid starvation EBSS +MEM Vitamins +10%FBS +P/S.

Growth protocol

HEK293 cells were cultured in DMEM supplemented with 10% FBS and P/S.

Extracted molecule

total RNA

Extraction protocol

Cells were washed and detached with 10 ml ice cold PBS and collected at 1000 rpm for 5 minutes at 4°C. Cells were lysed in 250 µl lysis buffer (100mM KCl, 20mM Tris pH 7.5, 5mM MgCl2, 1mM DTT, 1mM Leupeptin, 1mM Pepstatin, 1mM Aprotinin, 0,1 mM PMSF, 0,1 mM AEBSF, 0,5% NP40, 100 µg/ml cycloheximide, 40 U/ml RNAsin (Promega)) for 10 minutes on ice. Cell debris were pelleted at 10k rpm for 10 minutes in an Eppendorf tabletop centrifuge.
Ribosome protected fragments (between 17-34 nts) were purified on a 15% PAA 8M Urea page. First, the ends were repaired using T4 polynucleotide kinase (NEB) with and without ATP to create a phosphorylated 5’- and a dephosphorylated 3’-end for 1 h at 37 °C each. Afterwards, libraries were prepared using the NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (NEB) following the manufacturer’s protocol with only minor modifications. All adapters and primers were diluted 1:3 and 14 cycles of PCR were performed. No further size selection was performed. Libraries were quantified using a Qubit 3.0 (Thermo Scientific) and a 2100 Bioanalyzer (Agilent) and pooled equimolar. The pool of 12 ribosome profiling libraries was sequenced on a High-Output NextSeq 500 (Illumina) sequencing run with a read length of 75 nt.

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina NextSeq 500

Data processing

Briefly, after adapter trimming, reads were first mapped against an rRNA database using Bowtie (Version 1.0)
All remaining reads were mapped against the human genome and transcriptome (Ensembl v90). Read mappings were merged (keeping all mapping with minimal amount of mismatches). Price (Version 1.0.2) was used to map reads to P site codons.
Genome_build: HG38
Supplementary_files_format_and_content: Price (Erhard et al., Nature Methods 2018) output (identified and quantified ORFs)

Submission date

Apr 16, 2019

Last update date

Aug 04, 2022

Contact name

Florian Erhard

E-mail(s)

Florian.Erhard@uni-wuerzburg.de

Organization name

Julius-Maximilians-Universität Würzburg

Department

Institut für Virologie und Immunbiologie