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| Status |
Public on Apr 30, 2018 |
| Title |
RPF_sc_2 |
| Sample type |
SRA |
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| Source name |
ATCC Cell Lines
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| Organism |
Homo sapiens |
| Characteristics |
cell type: HeLa
treatment: scramble siRNA
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| Treatment protocol |
For siRNA transfection, 5 × 10^6 HeLa cells per 15 cm dish were cultured overnight and treated with 27 μL DharmaFECT 1 Transfection Reagent (Dharmacon) and treated with 90 nM indicated siRNA in 1.8 mL OptiMEM media.DHX9 and DHX36 siRNA oligoes were transfected at equimolar concentrations. A pool of four non-targeting siRNAs (ON-TARGETplus Non-targeting Control Pool, Dharmacon) was used as control. The cells were then expended for 48 h. For iCLIP, refer to associated publication.
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| Growth protocol |
HeLa cells were cultured in Dulbecco’s modified Eagle Medium (Life Technology) supplemented with 10% FBS (Life Technology).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA and ribosome-protected fragments (RPFs) were isolated using the TruSeq Ribo Profile Mammalian Kit (Illumina) following the manufacturer’s intructions with some modifications. For RPFs purification, lysates were treated with 5 U TruSeq Ribo Profile Nuclease per A260 of lysate. RPFs were isolated using Illustra MicroSpin S-400 HR Columns (GE Healthcare, 27- 5140-01). rRNAs were removed using the Ribo-zero Magnetic Gold kit (Illumina, MRZG12324). After PAGE purification (15% urea-polyacrylamide gel to select fragments ~28-30 nt in length), end-repair, 3’-adapter ligation, reverse transcription (EpiScript reverse transcriptase) and cDNA circularisation (CircLigase, Epicentre), RPFs were amplified using 9 PCR cycles using Phusion polymerase (NEB). For parallel RNA-seq, total RNA was processed similarly, but excluding the nuclease digestion and S-400 columns purification steps. Before the library preparation steps, total RNA samples were heat-fragmented (94°C for 25 minutes). PCR amplicons were purified on a 8% native polyacrylamide gel excising bands corresponding to ~70-80 nt and ~80-100 nt for RPF and total RNA samples respectively. Libraries were sequenced on a NextSeq 500 using 75-nt single-read sequencing runs. For iCLIP, refer to associated publication.
TruSeq Ribo Profile Mammalian Kit (Illumina)
RNA-Seq, Ribo-Seq, iCLIP-seq
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Ribosome protected RNA fragments
Ribo-seq
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| Data processing |
RNA-seq libraries were trimmed using cutadapt. The remaining reads were aligned to the GRCh38 human genome using the rsem-calculate-expression of RSEM
For all analyses the version 26 of the human genome transcript annotation from Gencode was used. For each experiment, the transcriptome was defined by considering transcripts with an expression above 1 TPM (Transcript Per Million) and isoform representation percentage above 5%.
22,911 transcripts representing the union transcriptome of all performed RNA-seq experiments were then considered for all following analyses, and the corresponding RPF libraries were aligned to it using RSEM. Only RPF reads in the size range 24-32 were considered for analysis.
Isoforms quantifications for both RNA-seq and RPF libraries were obtained in forms of estimated counts and TPM during the alignment procedure. Transcript coverage was calculated using RSEM command rsem-bam2wig and rsem-bam2readdepth, which both take into account multi-mapping reads and proportionally assign multiple assignments, a crucial step for processing short reads libraries.
For iCLIP analysis, refer to associated publicatoon.
Genome_build: GRCh38
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| Submission date |
Oct 17, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Giovanni Marsico |
| E-mail(s) |
persego@gmail.com
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| Organization name |
CRUK Cambridge Institute
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| Street address |
Robinson Way
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| City |
Cambridge |
| ZIP/Postal code |
CB2 0RE |
| Country |
United Kingdom |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE105082 |
RNA G-quadruplexes mark repressive upstream open reading frames in human mRNAs |
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| Relations |
| BioSample |
SAMN07795657 |
| SRA |
SRX3292090 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2817681_sc_2.isoforms.results.txt.gz |
1.1 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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