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| Status |
Public on Oct 23, 2015 |
| Title |
Lactimidomycin treated Jurkat cells |
| Sample type |
SRA |
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| Source name |
cell line
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| Organism |
Homo sapiens |
| Characteristics |
tissue: peripheral blood
cell_line: Jurkat T-lymphocytes
phenotype: Lymphoid-like
sample_type: cell culture
biomaterial_provider: ATCC
age: 14
Sex: male
cell_type: T Cell/ T Lymphocyte
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| Treatment protocol |
Cultures were treated with 50 µM lactimidomycin (LTM) (Ju et al, 2005; Schneider-Poetsch et al, 2010) or 100 µg/ml cycloheximide (CHX) (Sigma, USA) for 30 min at 37°C before cell harvest.
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| Growth protocol |
For ribosome profiling, Jurkat cells were grown in RPMI medium (Gibco) supplemented with 10% fetal bovine serum, 2 mM alanyl-L-glutamine dipeptide (GlutaMAX, Gibco), 50 units/ml penicillin and 50 µg/ml steptamycin at 37°C and 5% CO2.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were collected by centrifugation (5 min at 300g), rinsed with ice-cold PBS and recovered again by centrifugation in the presence of CHX. 108 cells were re-suspended in 1 ml ice-cold lysis buffer (10 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 100 mM KCl, 1% Triton X-100, 2 mM dithiothreitol (DTT), 100 µg/ml CHX, 1 × complete and EDTA-free protease inhibitor cocktail (Roche)) (Guo et al, 2010). Following 10 min of incubation on ice, samples were passed through QIAshredder spin columns (Qiagen). The flow-through was clarified by centrifugation for 10 min at 16,000 × g and 4°C. 600 µl of the recovered supernatant was subjected to RNase I (Life Technologies) digestion using 1000 U of enzyme. Digestion of polysomes proceeded for 55 min at room temperature and was stopped with SUPERase.In RNase Inhibitor (Life Technologies). Next, monosomes were recovered by ultracentrifugation at 75,000 RPM in a cooled TLA-120.2 rotor over a 1 M sucrose cushion in 20 mM HEPES-KOH pH 7.4, 5 mM MgCl2, 100 mM KCl, 2 mM DTT, 100 µg/ml CHX and 100 U/ml Superase.In. Subsequent steps were performed as described by Ingolia et al. (Ingolia et al, 2011) with some minor adjustments. RNA was extracted from the samples using a heated acid phenol; chloroform; isoamyl alcohol (125:24:1) procedure. 17 µg of RNA was subjected to electrophoresis under denaturing conditions in 15% TBE-Urea polyacrylamide gel (Life Technologies).
Ribosome protected fragments (RPFs) of 28-34 nucleotides were extracted from the gel in RNase-free water for 10 min at 70°C and precipitated with 1/10 volume of 3 M sodium acetate pH 5.2, 1.5 µl GlycoBlue (Life Technologies) and 1 volume of isopropanol overnight at − 80 °C. Following a dephosphorylation reaction (end-repair) with 10 U of T4 polynucleotide kinase (New England Biolabs), the RPFs were depleted of ribosomal RNA contaminants using the Ribo-Zero Magnetic Gold Kit (Illumina) according to the manufacturer’s protocol. Subsequently, fragments were ligated to a 1.5 µg of Universal cloning linker using 200 U of T4 RNA ligase II (both New England Biolabs). The ligated product was size-selected using denaturing gel electrophoresis and purified from gel as described above. Subsequently, reverse transcription (RT) was performed with 200 U of SuperScript III (Life Technologies) according to (Ingolia et al, 2011), followed by a size-selection of the RT product (described above). Next, samples were subjected to circular ligation using 100 U of CircLigase (Illumina) and amplified by PCR using Phusion polymerase (New England Biolabs) for 12 cycles of denaturation (10 s at 98°C), annealing (10 s of 65°C) and elongation (5 s at 72°C) using primer pairs compatible with the Illumina sequencing platform (Ingolia et al, 2011). The resulting ribosome-profiling libraries of LTM- and CHX- treated Jurkat cells were sequenced on a NextSeq 500 instrument (Illumina) to yield 75 bp single-end reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
ribosome-associated mRNA
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| Data processing |
Ribosome profiling (Ribo-Seq) performed in Jurkat cells treated with CHX to study translation elongation and LTM to study translation initiation were analysed using the PROTEOFORMER pipeline (Crappe et al, 2015). PROTEOFORMER was used to perform the quality control, mapping onto a reference genome, identification of TIS and generation of a protein database.
The 3’ adapter sequences were clipped using the fastx_clipper and reads shorter than 26 or longer than 34 nucleotides were discarded for the Ribo-Seq based TIS calling.
The reads were first mapped onto small nuclear RNA, tRNA and rRNA (parameter settings: -snRNA Y -tRNA Y –rRNA Y) sequences to eliminate contaminating reads.
The remaining reads were subsequently mapped onto the human GRCh37 reference genome (Ensembl annotation bundle 75) allowing for a maximum of 2 mismatches along the entire read. Reads mapping to a maximum of 16 locations on the genome were allowed and retained for every loci. All mapping steps were performed using STAR 2.4.0i.
RPF alignments were assigned to a specific P site nucleotide based on the length of the fragment. The offset from the 5’ end of the alignment was +12, +13 and +14 respectively for alignment of less than or equal to 30 bases long, 31–33 bases long, and greater than or equal to 34 bases long.
Genome_build: hg19
Supplementary_files_format_and_content: BedGraph: read alingments were reduced to single-nucleotide genomic position, based on P-site assignment. Separate BedGraph files were provided for the sense and antisense strand.
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| Submission date |
Oct 22, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Petra Van Damme |
| E-mail(s) |
petra.vandamme@ugent.be
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| Phone |
+3292649279
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| Organization name |
VIB
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| Street address |
Albert Baertsoenkaai 3
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| City |
Gent |
| ZIP/Postal code |
9000 |
| Country |
Belgium |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE74279 |
Positional proteomics reveals differences in N-terminal proteoform stability |
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| Relations |
| BioSample |
SAMN04195657 |
| SRA |
SRX1356474 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1916181_JURKAT_LTM_antisense.bedgraph.gz |
36.6 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM1916181_JURKAT_LTM_sense.bedgraph.gz |
36.9 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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