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Status |
Public on Sep 13, 2023 |
Title |
Wild-type male, RNAseq ZT02-04, rep2 |
Sample type |
SRA |
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Source name |
whole liver tissue
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Organism |
Mus musculus |
Characteristics |
tissue: whole liver tissue age: 12-14 week-old gender: male strain: C57BL/6N time: ZT02-04 genotype: wild-type
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Treatment protocol |
Half-dime size liver sections were snap-frozen and stored at -80C until use.
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Growth protocol |
Twelve-to-fourteen week-old wild-type male mice (C57BL/6N, Japan SLC) under 12-h light (400 lx) 12-h dark (LD) conditions were sacrificed between ZT02-04.
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Extracted molecule |
total RNA |
Extraction protocol |
Frozen liver sections were rinsed in 3x PBS, homogenized in 1 mL polysome lysis buffer using dounce A (10 strokes) then dounce B (10 strokes), clarified by 15,000 x g centrifugation for 10 min., then divided into 200 uL + 1 mL trizol (RNAsequencing), 300 uL for ribosome profiling (see steps below), and remaining snap frozen for western blot. Unbound RNA was digested by the addition of 7.5 µl RNAseIf (ThermoFisher) for 45 min and then stopped by 10 µl SUPERase In RNAse Inhibitor (ThermoFisher). The digestion was transferred to 13 mm × 51 mm polycarbonate ultracentrifuge tubes, layered on top of 0.9 ml sucrose cushion (150 mM NaCl, 20 mM Tris-HCl pH 7.4, 5 mM MgCl2, 5 mM DTT, 100 µg/ml cycloheximide, 1 M sucrose, 10 U/ml SUPERase In), centrifuged in a TLA100.3 rotor at 70,000 rpm at 4 °C for 4 h. Ribosome pellets were resuspended in 0.8 ml Trizol and mRNA was recovered according to the manufacturer's instructions.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NovaSeq 6000 |
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Description |
M3W_RNA_S2 total RNA
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Data processing |
RNA was precipitated from the elution by the addition of 38.5 µl water, 1.5 µl GlycoBlue (ThermoFisher), and 10 µl 3 M sodium acetate pH 5.5 followed by 150 µl isopropanol, stored overnight at -80 °C, pelleted by centrifugation (20,000 × g for 30 min at 4 °C), and resuspended in 22 µl 10 mM Tris pH 8. Approximately 10 µg RNA was separated on a 15% (wt/vol) polyacrylamide TBE-urea gel at 200 V for 65 min in 1× TBE, and fragments between 26 and 34 nt were excised and recovered in 400 µl RNA gel extraction buffer (300 mM sodium acetate pH 5.5, 1 mM EDTA, and 0.25% (wt/vol) SDS) overnight. RNA was precipitated by the addition of 1.5 µl GlycoBlue and 500 µl, stored at -80 °C for >30 min, pelleted by centrifugation (20,000 × g for 30 min at 4 °C), and resuspended in 10 µl 10 mM Tris pH 8. RNA was dephosphorylated by T4 PNK (Takara Bio), precipitated and recovered as previously described, resuspended in 8.5 µl 10 mM Tris, and 1.5 µl of preadenylated and 3' blocked miRNA cloning linker (1/5rApp/CTGTAGGCACCATCAAT/3ddC/, IDT) was added. The linker mixture was denatured for 90 s at 80 °C, cooled to room temperature, ligated by T4 Rnl2 (NEB) for ~24 h at room temperature, precipitated, recovered, and separated by 15% TBE-urea gel electrophoresis. Ligation products were recovered overnight in RNA gel extraction buffer, precipitated, and recovered in 10 µl 10 mM Tris pH 8. Ligated RNA was reverse transcribed at 52 °C for 20 min using SuperScript IV (ThermoFisher) with the reverse transcription primer (5′-(Phos)-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGC-(SpC18)-CACTCA-(SpC18)-TTCAGACGTGTGCTCTTCCGATCTATTGATGGTGCCTACAG-3′, where SpC18 indicates a hexa-ethyleneglycol spacer). Reverse transcription products were precipitated, recovered, separated by 15% TBE-urea gel electrophoresis, and recovered overnight in DNA gel extraction buffer (300 mM NaCl, 10 mM Tris pH 8, and 1 mM EDTA). The cDNA was precipitated, recovered, resuspended in 15 µl 10 mM Tris pH 8, and circularized by CircLigase (EpiCentre) for 1 h at 60 °C. rRNA was depleted by combining 5 µl circularization reaction with 1 µl of subtraction oligo pool, 1 µl 20× SSC, and 3 µl water, incubating at 37 °C for 15 min and binding to MyOne Streptavidin C1 DynaBeads (ThermoFisher) at 37 °C for 15 min with mixing at 1000 rpm. Eluate was precipitated, recovered, and resuspended in 5 µl 10 mM Tris pH 8. rRNA-depleted cDNA was PCR amplified by Phusion High-Fidelity DNA Polymerase (NEB) in a 20 µl reaction (denature 94 °C for 15 s, anneal 55 °C for 10 s, extend 72 °C for 10 s) for 12 cycles. PCR products were separated on a 8% TBE gel, recovered overnight in DNA gel extraction buffer, precipitated, recovered, and resuspended in 15 µl 10 mM Tris pH 8. Libraries were quantified using the high-sensitivity DNA chip on the Agilent BioAnalyzer according to the manufacturer's protocol, pooled, and amplified on the Illumina NovaSeq6000 system according to the manufacturer's protocol. RNAseq libraries were amplifed using the TruSeq stranded mRNA kit according to the manufacturer's instructions. For RNAseq analysis, the data were processed using DESeq2. For ribosome profiling, the follow steps were taken: after quality control using FASTQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/), reads were clipped using fastx_clipper (with parameters -Q33 -a CTGTAGGCACCATCAAT -l 25 -c -n) and processed with fastx_trimmer (with parameters -Q33 -f 2), both from the FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/). We then used Bowtie 2.1.0.0 (Langmead and Salzberg, 2012) to map the resulting reads to rRNA sequences. Reads that were successfully aligned to these sequences are discarded, and only unaligned reads were used for downstream analysis. These reads that did not align to rRNA were mapped to the mouse mm10 reference genome. Using samtools 0.1.19.0 and egrep, we extracted reads for which an exact match between the sequence and the reference could be found. Using reads aligned to a unique location, we then calculated transcript density profiles. This relies on assignment each footprint alignment (i.e., each read) to a specific A site nucleotide based on the length of the fragment. The initial assumption is that this site will be close to the center of the read. To calculate the best offset, we considered a metagene that captured all reads and their position relative to the start site and calculated the offset that optimized phasing. The best results were obtained when the offset from the 5' end of the alignment was: 26 nt long, +13; 27-28 nt long, +15; 29-30 nt long, +16; 31-32 nt long, +17. Reads shorter than 26 or longer than 32 nucleotides were discarded. Using this offset, we assigned each read to a unique nucleotide, and constructed transcript density profiles by counting the number of reads whose A site was assigned to each nucleotide position. For each transcript, non-overlapping five-codon windows were then tiled across the coding region and the transcript was considered well-translated if it had a median value of at least 2 reads per window (excluding the excluding the first fifteen and the last five codons).Using all well-translated transcripts, we created file where each transcript is represented across all samples by its RPKM value. We used this file in JTK_CYCLE (Hughes et al., 2010) to identify all rhythmic transcripts with a circadian period (i.e., period between 20 and 28 h). We used a threshold of 0.05 on the adjusted p-value to assess significance. Assembly: mm10
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Submission date |
May 05, 2023 |
Last update date |
Sep 13, 2023 |
Contact name |
Arthur Millius |
E-mail(s) |
arthur.millius@ifrec.osaka-u.ac.jp
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Phone |
+81-06-6879-8366
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Organization name |
Osaka University
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Department |
iFReC
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Lab |
Systems Immunology
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Street address |
iFReC 6F
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City |
Suita |
State/province |
Osaka |
ZIP/Postal code |
565-0871 |
Country |
Japan |
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Platform ID |
GPL24247 |
Series (1) |
GSE231820 |
Circadian ribosome profiling reveals a role for the Period2 upstream opening reading frame in sleep. |
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Relations |
BioSample |
SAMN34722040 |
SRA |
SRX20238441 |
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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