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| Status |
Public on Feb 10, 2017 |
| Title |
HEK293T tRNA Treated |
| Sample type |
SRA |
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| Source name |
HEK293T
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| Organism |
Homo sapiens |
| Characteristics |
treatment: AlkB-derived enzyme mixture
cell line: HEK293T
molecule: tRNA
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| Treatment protocol |
Human HEK293T cell line was cultured in DMEM (Thermo) media supplemented with 10% FBS and 1% 100 × Pen Strep (Gibco)
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by using mirVana miRNA Isolation Kit (Life Technologies). Purified total RNA was deacylated by incubating in 0.1 M tris-HCl, pH 9 at 37°C for 45 min. When necessary, total tRNA was subsequently isolated using a denaturing 10% polyacrylamide gel followed by passive gel elution and ethanol precipitation.
Homemade protocol based on Illumina protocols. Deacylated RNAs with or without the demethylation were first treated with T4 Polynucleotide Kinase (Epicentre) at 37C° for 30 min to further warrant a free 3' hydroxyl group for template switching. Template-switching reactions were performed as described7. Briefly, we used an initial template-primer substrate consisting of a 41-nt RNA oligonucleotide (5’-AGA UCG GAA GAG CAC ACG UCU AGU UCU ACA GUC CGA CGA UC/3SpC3/-3’) that contains Illumina Read1 and Read2 primer-binding sites and a 3’ blocking group (three carbon spacer; IDT) annealed to a complementary 32P-labeled DNA primer with a single-nucleotide 3’ overhang, T, which facilitates the template switch to full-length tRNA that mostly contain a 3’ CCA end. For g2RT template-switching reactions, average 100 ng of demethylated tRNAs or 1 μg of demethylated total RNA were mixed with the initial template-primer substrate (100 nM) and g2RT (1 μM of TeI4cΔEn or 500 nM of GsI-IIC RT) in reaction medium containing 450 mM NaCl, 5 mM MgCl2, 20 mM Tris-HCl, pH 7.5, and DTT (1 mM for TeI4cΔEn or 5 mM for GsI-IIC RT). For template-switching with TeI4cΔEn RT, the reactions were pre-incubated at room temperature for 30 min, initiated by adding 25 mM dNTPs to a final concentration of 1 mM and incubating at 60°C for 30 min. For template-switching with GsI-IIC RT, the pre-incubation step is not required. The reactions were terminated by adding 5 M NaOH to a final concentration of 0.25 M, incubating at 95°C for 3 min, and neutralizing with 5M HCl. The cDNAs resulting from template switching were analyzed in a denaturing 6% polyacrylamide gel, electroeluted using a D-tube Dialyzer Maxi with MWCO of 6-8 kDa (EMD Millipore), and ethanol precipitated with 0.3 M sodium acetate in the presence of 25 µg of linear acrylamide (Life Technologies). The purified cDNAs were then circularized with CircLigase II (Epicentre) using manufacture’s protocol with extended incubation time of 5 hours at 60°C, phenol-CIA extracted, ethanol precipitated and amplified with Phusion-HF (Thermo Scientific) using Illumina multiplex (5’- AAT GAT ACG GCG ACC ACC GAG ATC TAC ACG TTC AGA GTT CTA CAG TCC GAC GAT C -3’) and barcode (5’- CAA GCA GAA GAC GGC ATA CGA GAT BARCODE GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T -3’) primers for 12 cycles of 98°C for 5 sec, 60°C for 10 sec and 72°C for 10 sec. The PCR products were sequenced on Illumina HiSeq 1000 system.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 1000 |
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| Description |
library strategy: DM-tRNA-Seq
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| Data processing |
Raw reads were processed using Trimmomatic v 0.32n and custom Python scripts to fully remove traces of adapter.
Processed reads were aligned in Bowtie 1.0 according to the parameters of -t -v 3 -m 10 --best --strata -p 12
Fragments shorter than 18 base pairs during alignment were removed during subsequent analysis.
Further processing using custom C and Python scripts were used to map reads onto tRNA isodecoders/rRNAs for visualization and modification index analysis.
Genome_build: hg19
Supplementary_files_format_and_content: Individual alignment files mapping tRNA to isodecoder with read counts
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| Submission date |
Dec 30, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Tao Pan |
| E-mail(s) |
taopan@uchicago.edu
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| Phone |
(773) 702-4179
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| Organization name |
University of Chicago
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| Department |
Biochemistry and Molecular Biology
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| Street address |
929 E. 57th Street
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| City |
Chicago |
| State/province |
Illinois |
| ZIP/Postal code |
60637 |
| Country |
USA |
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| Platform ID |
GPL15433 |
| Series (1) |
| GSE76434 |
tRNA Modification Identification and Quantification through High Throughput Sequencing |
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| Relations |
| BioSample |
SAMN04377916 |
| SRA |
SRX1509042 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2011597_DemethylaseTreatedtRNAMap293T.txt.gz |
6.4 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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