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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Sep 30, 2021 |
| Title |
neural tube, Mutant Ribo-seq (run2_TC5_mut_Ribo_S5_L004) |
| Sample type |
SRA |
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| Source name |
neural tube
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| Organism |
Mus musculus |
| Characteristics |
age: E11.5
genotype: C3HeB/Eif3cXs-J/+
tissue: neural tube
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| Extracted molecule |
total RNA |
| Extraction protocol |
Ribosome profiling was performed following the procedures described before (Fujii et al., 2017). For Ribo-Seq, neural tubes from 1 litter of E11.5 embryos (20-23 somite number from tail to hind limb) were used. The Papain dissociated cells were split into two tubes. One was dissolved by Trizol (Invitrogen, 15596) for RNA seq. Another cell pellet was re-suspended in 0.5 ml of cold lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 15 mM MgCl2, 1 % Triton X-100, 1 mM DTT, 8 % Glycerol, 20 Unit/ml TURBO DNase, 100µg/ml Cycloheximide) and incubated for 30 min at 4 °C with occasional vortexing. The lysate was clarified by sequential centrifugation for 5 min at 1,800 g and 10,000 g at 4 °C to remove nuclei and mitochondria. The lysate was then treated with RNase I (Ambion, AM2294) for 30 min at RT to digest mRNAs not protected by the ribosome. The digestion was stopped by adding 4.5 µl of SUPERaseIn RNase Inhibitor (20 U/µl, Ambion AM2696). Lysate was then loaded onto a 1 M sucrose cushion. Ribosomes and ribosome protected fragments (RPFs) were pelleted by ultracentrifugation at 70,000 rpm for 4 hr at 4 ºC by TLA120.2 rotor. The pellet was re-suspended in Trizol (Invitrogen, 15596) and RNAs were extracted by following manufacturer’s protocol. RNA-seq: RNA was extracted from Trizol (Invitrogen, 15596) following manufactuer’s protocol and polyA mRNA was isolated using Oligotex mRNA Mini Kit (Qiagen, 70022) following manufacturer’s protocol. Purified mRNAs were fragmented in alkaline fragmentation buffer (100 mM NaCO3 pH 9.2, 2 mM EDTA).
Ribo-seq/RNA-seq Library production: RPFs and fragmented RNAs were loaded onto a 15% urea gel. 28–31 nt RPFs and 30–50 nt fragmented RNAs were excised from the gel for Ribo-Seq and RNA-Seq respectively. RNAs were eluted, dephosphorylated by PNK (NEB, M0201S), and ligated to the miRNA Cloning linker (NEB, S1315S) by T4 RNA Ligase2 truncated K227Q (NEB, M0242S). Ligated RNA was gel purified and reverse transcribed by Superscript III (Invitrogen, 18080). Gel purified cDNAs were circularized by Circligase (Epicentra, CL4111K) and rRNA sequence were subtracted using biotinylated oligos. Amplification was done using Phusion High Fidelity DNA Polymerase (NEB, M0530S). PCR amplification was performed for 6–7 cycles and products were loaded onto non-denaturing 8 % PAGE gel. DNA fragment were purified for Illumina sequencing.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Replicate 2
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| Data processing |
Library strategy: Ribo-seq
Read alignment: For removal of adapter sequences, low quality bases, and short reads, we use cutadapt (Martin, 2011) to trim Illumina adapter sequences and <Q20 bases (parameters -m 18 -a CTGTAGGCACCATCAAT --quality-cutoff=20). For splice-aware alignment using STAR (Dobin et al., 2013), we used STAR to align the reads to a reference genome/transcriptome. STAR reference is built using a combination of mouse genome (mm10), mouse rDNA sequence (GenBank GU372691), and mouse transcript annotations (GENCODE vM18). Only uniquely mapped reads were retained (Parameters --outFilterMultimapNmax 1 --alignEndsType EndToEnd --alignIntronMax 1000000 --alignIntronMin 20 --outFilterMismatchNmax 999 --alignSJDBoverhangMin 1 --alignSJoverhangMin 8). For read quantification, we used bedtools (Quinlan and Hall, 2010) to count alignments over annotated coding regions excluding the first 45 bases from the start codon and last 15 bases from the stop codon.
Genome_build: STAR reference is built using a combination of mouse genome (mm10), mouse rDNA sequence (GenBank GU372691), and mouse transcript annotations (GENCODE vM18).
Supplementary_files_format_and_content: Text file includes read counts across the different regions of each annotated transcript
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| Submission date |
Sep 05, 2021 |
| Last update date |
Sep 30, 2021 |
| Contact name |
Gun Woo Byeon |
| Organization name |
Stanford University
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| Street address |
279 Campus Dr., Beckman Center B309
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| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE183470 |
Controlling tissue patterning by translational regulation of signaling transcripts through the core translation factor eIF3c [I] |
| GSE183472 |
Controlling tissue patterning by translational regulation of signaling transcripts through the core translation factor eIF3c |
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| Relations |
| BioSample |
SAMN21238194 |
| SRA |
SRX12020239 |
| 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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