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| Status |
Public on Oct 21, 2015 |
| Title |
Flag-DKC1 |
| Sample type |
SRA |
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| Source name |
293FT-Flag-DKC1
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293
cell type: human embryonal kidney cells
genotype/variation: Flag-DKC1 with tetracycline-sensitive promoter
induced with: 500 ng/ml doxycycline
rip antibody: Anti-Flag M2
rip antibody vendor: Sigma-Aldrich
rip antibody cat.#: A2220
rip antibody lot #: SLBJ3073V
molecule subtype: human telomerase RNA
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| Treatment protocol |
Expression from the tetracycline-sensitive promoter was induced using 500 ng/ml doxycycline.
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| Growth protocol |
Cells were grown in DMEM containing 10% tetracycline-free FBS, Hygromycine (75 µg/ml), and Blasticidin (15 µg/ml), and were passaged until they reached 90% confluency.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA co-immunoprecipitation (RIP) assays and extraction of RNA from beads were carried out as previously described (Bergeron et al. 2015, Mol. Cell. Biol., 35: 2503-2517).
Preparation of human telomerase RNA-specific libraries was based on the RNA ligase-mediated 3’ RACE approach described by Goldfarb and Cech (BMC Mol Biol. 2013, 14: 23). Barcoded libraries were combined and analyzed on an Illumina MiSeq instrument.
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
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| Data processing |
Cutadapt was used to remove tags and adaptors from the raw reads obtained from the fastq files for each sequencing experiment. Because read lengths are longer than the RNAs sequenced, adapters are present at both ends of the reads. Cutadapt was thus run twice to remove adapters at both ends, with parameter values -g CTGGAATTCGCGGTTAAA -a ANANCGGAANANCACACGTCTGAACTCCAGTCNC for the first run and -g TGGNGTTNGACGTGTGCTCTTCCGATCT -a TTTANCCGCGAATTCCAN for the second run. Other parameters were set to --minimum-length 10 --match-read-wildcards --times 5 -q 20 --paired-output.
The resulting reads were aligned using Bowtie2 to an index consisting of all non-coding RNAs annotated on build hg38 of the human genome by Ensembl. Bowtie parameter values used were –local, -I 13.
The resulting SAM file was processed using a custom python script which captures a regular expression present in the hTR sequences, namely the ACA box found 3 residues from the 3’ end of hTR, and calculates the number of non-genomic consecutive As following the 3’ end of the molecule. Using this strategy, the script counts the number of reads aligned to hTR, not aligned to hTR, having less than 3 nucleotides between the 3’ end and the ACA box, and the number of reads for each length of polyadenylated tail.
Genome_build: hg38
Supplementary_files_format_and_content: Excel file compiling the abundances of all forms of hTR
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| Submission date |
Oct 20, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Michelle S Scott |
| E-mail(s) |
[email protected]
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| Organization name |
University of Sherbrooke
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| Department |
Biochemistry
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| Street address |
3201 Jean Mignault
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| City |
Sherbrooke |
| State/province |
Quebec |
| ZIP/Postal code |
J1E 4K8 |
| Country |
Canada |
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| Platform ID |
GPL15520 |
| Series (1) |
| GSE74186 |
Next generation sequencing identifies polyadenylated species of hTR |
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| Relations |
| BioSample |
SAMN04196539 |
| SRA |
SRX1356970 |
| 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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