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Status |
Public on Jun 23, 2021 |
Title |
LPS15_RPF |
Sample type |
SRA |
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Source name |
RAW264.7
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Organism |
Mus musculus |
Characteristics |
cell line: RAW264.7 material: ribosome-protected fragments treatment: LPS, 15 min
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Treatment protocol |
RAW264.7 macrophages were stimulated with 100 ng/ml LPS (E. coli O111:B4, Sigma L2630) for the indicated time-points.
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Growth protocol |
RAW264.7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS, Biochrome), 2 mM L-Glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin (all PAN Biotech) at 37°C in a humidified atmosphere with 5% CO2. Every two to three days, cells were carefully detached by scraping in warm 1x PBS and diluted 1:10. On the day before experiments, RAW264.7 macrophages were seeded at a density of 1.2 x 107 cells per 10 cm dish (Sarstedt, 83.3902).
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Extracted molecule |
cytoplasmic RNA |
Extraction protocol |
Cells were washed once in ice-cold 1 x PBS supplemented with 100 µg/ml cycloheximide (Roth, 8682.3), and harvested by scraping in polysome lysis buffer (20 mM Tris-HCl pH 7.4, 10 mM MgCl2, 200 mM KCl2, 1% NP-40, 100 µg/ml cycloheximide, 2 mM DTT, 1 tablet EDTA-free Roche cOmplete Mini Protease Inhibitor per 10 ml). Lysates were rotated end-over-end for 10 min at 4°C and cleared by centrifugation at 9,300 g for 10 min at 4°C. About 10% of the lysates were saved as input samples. The lysates were subsequently digested with RNase I (240 U per A260; Ambion AM2294) for 5 min at 4°C. Samples were then subjected to 17.5–50% sucrose density gradient centrifugation (for 1 h 45 min at 40,000 rpm and 4°C in a SW60 rotor) and fractions of 250 – 300 µl were collected with a Teledyne Isco Foxy Jr. fractionator directly into 300 µl urea buffer (10 mM Tris-HCl ph 7.5, 350 mM NaCl, 10 mM EDTA, 1% SDS, 7 M urea). RNA was purified from the cytoplasmic lysate (input) or from the monosomal fractions (ribosome footprint) using phenol:chloroform:isoamylalcohol (25:24:1, AppliChem A0944) by phase separation and precipitation with GlycoBlue (Ambion AM9515) in 50 – 60% isopropanol. Both input and ribosome protected fragments were depleted of ribosomal RNA (rRNA) with the Ribo-Zero Gold Kit (Illumina MRZG126). Input RNA was randomly fragmented by alkaline hydrolysis at pH 10.0 for 12 min at 95°C. Fragmented RNA and ribosome protected fragments were size-selected (25 – 35 nt) on a 15% polyacrylamide Tris-borate-EDTA-urea gel after staining with SybrGold. RNA was eluted from the gel slices by rotating at 4°C overnight in 300 mM NaCl plus RNase OUT (Invitrogen). The gel matrix was removed by centrifugation in a 0.45 µM NanoSep MF tube (PALL). After precipitation with isopropanol and GlycoBlue, end-repair was performed with 10 U T4 PNK (NEB M0201S), 40 U RNase OUT and 1 mM ATP in T4 PNK reaction buffer for 1 h at 37°C. After end-repair with T4 PNK, libraries were prepared with the NEXTflex Small RNA-Seq Kit v3 according to the manufacturer’s manual, using 2 ng RNA. In order to determine the number of PCR cycles required to obtain a ~ 20 nM library, 1 µl of cDNA per sample was diluted 1:8 and used for a SybrGreen qPCR (forward primer: 5’-GTTCAGAGTTCTACAGTCCGA-3’, reverse primer: 5’-CCTTGGCACCCGAGAATTCCA-3’, SybrGreen master mix: applied biosystems A25742) on a QuantStudio Real-Time PCR System. For library preparation, three cycles less than the highest determined cycle of threshold were used (14 cycles).
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NextSeq 500 |
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Data processing |
Adapters were removed with the FASTX-toolkit (http://hannonlab.cshl.edu/fastx_toolkit/), retaining only sequences that are at least 28 nt long. The four random nucleotides at the beginning and the end of the reads were trimmed with an in-house developed perl script. Alignment was performed with bowtie v0.12.8 (Langmead et al., Genome Biology 2009) allowing a maximum of two mismatches and reporting all alignments in the best stratum (settings: -a –best –stratum –v 2). Reads that did not map to tRNA or rRNA sequences (as downloaded from the UCSC Genome Browser) were aligned to a mouse transcriptome (Gencode VM18 as downloaded from the UCSC Genome Browser wgEncodeGencodeBasicVM18 table). Reads were filtered with in-house developed perl scripts to retain only reads that are between 25 and 35 nt long and map to ORFs of isoforms arising from one specific gene (as defined by a common gene symbol). An offset of 12 nt upstream of the start codon and 15 nt upstream of the stop codon with respect to the 5’end of the read was assumed. We resampled reads that could be uniquely assigned to ORFs to obtain exactly the same number of reads per sample (~ 6.7 million), in order to avoid different degrees of noise due to sampling error between IN and FP samples as well as between different time-points Normalization was performed using size factors obtained with the median ratio method of the DeSeq2 package, v1.18.1 (Love et al., Genome Biology 2014). Genome_build: mm10 Supplementary_files_format_and_content: Raw read counts as well as DESeq2-normalized expression values
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Submission date |
Jul 27, 2020 |
Last update date |
Jun 23, 2021 |
Contact name |
Johanna Daniela Schott |
E-mail(s) |
johanna.schott@medma.uni-heidelberg.de
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Organization name |
Heidelberg University
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Department |
Mannheim Institute for Innate Immunoscience
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Lab |
Stoecklin lab
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Street address |
Ludolf-Krehl-Str. 13-17
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City |
Mannheim |
ZIP/Postal code |
68167 |
Country |
Germany |
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Platform ID |
GPL19057 |
Series (2) |
GSE155233 |
Ribo-Seq timecourse in RAW264.7 cells upon LPS treatment |
GSE155236 |
ActD, Ribo-Seq and nRibo-Seq timecourses in RAW264.7 cells and mESCs |
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Relations |
BioSample |
SAMN15654984 |
SRA |
SRX8833507 |
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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