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| Status |
Public on Apr 23, 2021 |
| Title |
In-line-seq |
| Sample type |
SRA |
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| Source name |
in vitro transcribed
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| Organism |
synthetic construct |
| Characteristics |
tissue: in vitro transcribed
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| Extracted molecule |
total RNA |
| Extraction protocol |
Quenched RNA samples were purified with 2 µL of Oligo dT bead (Thermo Fisher, AM1922) and 0.8 µL of 10 µM FAM-A20-Tail2. RNA, bead, and primer were incubated at room temperature for 15 min, pulled down by magnetic stand for 10 min, and washed twice with 70% ethanol and left to dry on the bead. RNA was eluted in 2.5 µL of nuclease-free water (RNA was binded with OligodT bead and FAM.A20 Tail2 at this point)
5 µL of purified RNA was added to a reaction mixture containing 1x First Strand buffer (Thermo Fisher), 5 mM dithiothreitol (DTT), 0.8 mM dNTPs, 0.6 µL of SuperScript III RTase (Thermo Fisher) to a final volume of 15 µL. The reaction was incubated at 48 °C for 40 minutes and stopped with 5 µL of 0.4 M sodium hydroxide. The reaction was then incubated at 90 C for 3 minutes, cooled on ice for 3 minutes, and neutralized with 2 µL of quench mix (prepared as 2 mL of 5 M sodium chloride, 3 mL of 3 M sodium acetate, 2 mL of 2 M hydrochloric acid). cDNA was pooled down with 1.5 uL of Oligo C’ beads64 (in house magnetic beads prepared by immobilizing 2x Biotin oligonucleotides with Dynabeads™ MyOne™ Streptavidin C1; Thermo Fisher Scientific 65001), washed twice with 70% ethanol, then resuspended in 3.0 µL of water. We pooled 1.5 µL of each sample together, and took 9 µL of cDNA to continue to ligation an Illumina adapter by using Circ. Ligase I (Lucigen) at 68°C for 2 h. The reaction was stopped by incubation at 80 °C for 10 min. cDNA was added to 10 ul of 5 M NaCl and pulled down with a magnetic stand and washed with 70% ethanol; the ligated product was resuspended in 15 µL H2O.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
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| Data processing |
Library strategy: Barcode amplicon sequencing
Sequencing reads were analyzed using MAPseeker (https://ribokit.github.io/MAPseeker) following the recommended steps for sequence assignment, peak fitting, background subtraction of the no-modification control, correction for signal attenuation, and reactivity profile normalization.
processed data files format and content: Information about the degradation rates and reactivity rates of each nucleotide in each construct in the library are provided in RDAT format (https://rmdb.stanford.edu/deposit/specs/), a standardized format for reporting construct sequences, experimental conditions, and per-nucleotide signal for nucleic acid probing experiments.
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| Submission date |
Apr 22, 2021 |
| Last update date |
Apr 23, 2021 |
| Contact name |
Rhiju Das |
| E-mail(s) |
rhiju@stanford.edu
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| Organization name |
Stanford University School of Medicin
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| Department |
Biochemistry
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| Lab |
Das
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| Street address |
279 Campus Dr, B419 Beckman Center
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| City |
Stanford |
| State/province |
California |
| ZIP/Postal code |
94305 |
| Country |
USA |
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| Platform ID |
GPL27609 |
| Series (1) |
| GSE173083 |
Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics |
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| Relations |
| BioSample |
SAMN18828582 |
| SRA |
SRX10656880 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5259588_RYOS1_50C_0000.rdat |
4.8 Mb |
(ftp)(http) |
RDAT |
| GSM5259588_RYOS1_MG50_0000.rdat |
4.8 Mb |
(ftp)(http) |
RDAT |
| GSM5259588_RYOS1_MGPH_0000.rdat |
4.8 Mb |
(ftp)(http) |
RDAT |
| GSM5259588_RYOS1_NMD_0000.rdat |
15.1 Mb |
(ftp)(http) |
RDAT |
| GSM5259588_RYOS1_PH10_0000.rdat |
4.8 Mb |
(ftp)(http) |
RDAT |
| GSM5259588_SHAPE_RYOS_0620.rdat |
5.2 Mb |
(ftp)(http) |
RDAT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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