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| Status |
Public on Nov 21, 2017 |
| Title |
HeLa-experimental |
| Sample type |
SRA |
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| Source name |
HeLa cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
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| Treatment protocol |
Induction of LRPPRC-FLAG expression was achieved by addition of 2 μg/ml doxycycline to the incubation medium for 72 h.
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| Growth protocol |
HeLa Tet-On cells stably transformed with pTRE-hLRPPRC-FLAG were generated as previously described (Ruzzenente et al., EMBO J, 2012) and maintained in DMEM medium supplemented with GlutaMAX-I, 10% fetal bovine serum, 1% penicillin-streptomycin and 200 μg/ml hygromycin (Harmel et al., J Biol Chem, 2013).
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| Extracted molecule |
total RNA |
| Extraction protocol |
PAR-CLIP was performed according to Spitzer et al., 2013, with the exception that cells were lysed in lysis buffer (10 mM Tris-HCl (pH 7.5), 260 mM sucrose, 100 mM KCl, 20 mM MgCl2, 1% digitonin) in the presence of RNase inhibitor (New England Biolabs) and protease inhibitors (Roche). Lysates were clarified by centrifugation and diluted in lysis buffer without digitonin until the final digitonin concentration was 0.2%. Diluted lysates were incubated with anti-FLAG M2 magnetic beads (Sigma, Cat. No. M8823) for 2.5 h at 4°C. Beads were washed in lysis buffer with 0.1% digitonin and then in lysis buffer without digitonin.
RNA library preparation and sequencing was performed by Vertis Biotechnologie (Freising, Germany) using unique molecular identifiers to identify PCR duplicates according to Kivioja et al., 2011.
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Adapter sequences were removed from PAR-CLIP reads with cutadapt v1.10, requiring a minimum length after trimming of 14 nt.
Trimmed reads were aligned to the Numts-masked genome sequence with Bowtie v1.12 (-k 1 -n 2 -M 100 --chunkmbs 512 --best --strata).
Strand-specific coverage profiles were generated with bedtools v2.26.0
Genome_build: hg38
Supplementary_files_format_and_content: Strand-specific bedGraph files of mitochondrial read coverage
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| Submission date |
Jul 03, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Stefan J Siira |
| Organization name |
The Kids Research Institute Australia
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| Department |
Precision Health
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| Lab |
Mitochondrial Medicine and Biology
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| Street address |
15 Hospital Ave, Nedlands
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| City |
Perth |
| State/province |
Western Australia |
| ZIP/Postal code |
6009 |
| Country |
Australia |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE100730 |
LRPPRC-mediated folding of the mitochondrial transcriptome [PAR-CLIP HeLa] |
| GSE100733 |
LRPPRC-mediated folding of the mitochondrial transcriptome |
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| Relations |
| BioSample |
SAMN07311917 |
| SRA |
SRX2978826 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2692141_HeLa.fwd.chrM_coverage.bedGraph.gz |
97.1 Kb |
(ftp)(http) |
BEDGRAPH |
| GSM2692141_HeLa.rev.chrM_coverage.bedGraph.gz |
65.5 Kb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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