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| Status |
Public on Aug 26, 2022 |
| Title |
Polysomal_RNA_sequencing_Control_1.fastq.gz |
| Sample type |
SRA |
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| Source name |
HeLa cells
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| Organism |
Homo sapiens |
| Characteristics |
treatment: Control Polysomal RNA sequencing
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| Growth protocol |
HeLa, U2OS, HEK293 and MEF cell lines were grown in Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C. Cell lines were regularly tested for Mycoplasma contamination. Cell lines were authenticated by expression analysis based on RNA-seq.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Polysomal RNA isolation was performed as described previously (Slobodin et al., 2017). Briefly, Sucrose gradients for separation of polysomes were usually prepared by gentle sequential addition of 2.2ml of the different sucrose solutions (e.i., 47, 37, 27, 17 and 7% in Tris-HCl pH = 7.5 (f.c. 20mM), MgCl2 (f.c. 10mM) and KCl (f.c. 100mM), supplemented with 2mM DTT, Ribosafe RNase inhibitor (Bioline, 1 μl/ml) and CHX (100 μg/ml) into a 12 mL tube (Beckman, 9/16 × 3 1/2 in.) and left overnight at 4°C to achieve continuous gradient prior to the centrifugation. Cells were treated with CHX and harvest after washing with PBS with CHX and lysed. The lysates were centrifuged 1300xg for 10 min at 4°C and the supernatants were transferred into new tubes. From the cleared lysates, 500 μL were loaded on top of each gradient, mounted on SW41TI rotor and centrifuged at 36000rpm for 2 hr at 4°C. Following the centrifugation, each gradient was split into 15 equal fractions of 760 μl. Fractions 9-13 were collected for RNA isolation using TRIsure (Bioline) according to the manufacturer’s instructions, polyA selected followed by RNA library preparation as previously describe (Takara; 634839).
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
RNA-seq (as well as 3’, 5’, Polysome): FASTQ files were aligned to the human hg19 genome using either STAR (STAR_2.5.0a) or TopHat (v.2.0.13). Mouse FASTQ files were alignes with the same tools to the mm10 genome. TopHat aligned reads were aligned allowing up to 3 mismatches per read, a maximum gap of 5 bases, and a total edit distance of 8. Peaks were called using mapped reads from all samples, using findPeaks (Homer package, v.3.12) witth “-region -size 200 -minDist 250 -strand” parameters. Peaks found within transcripts or up to 5 kb downstream of promoters were included in a GTF file and processed by the DESeq package. Aligned Reads on exons were counted using HTSEQ (0.5.4p3).
Ribo-seq: Fastq file preprocessing consisted of adapter removal using cutadapt using the parameters (--quality-base=33 -O 7 -e 0.15 -m 20 -q 5) and removal of rRNA and tRNA contaminants by means of alignment against a reference using bowtie2with parameters (--seed 42 -p1 –local). The actual alignment of pre-processed fastq files was done with tophat2 and bowtie2 against GRCh37/hg19 using parameters ( --seed 42 -n 2 -m 1 --no-novel-juncs --no-novel-indels --no-coverage-search --segment-length 25). In a subsequent step the primary aligned reads filtered for a minimum mapping quality of 10. HTSEQ was used to count reads at exons of genes. Normalization was done using DESEQ.
3‘ end sequencing: same as Rnaseq with additionally removal of polyA sequences from the 3′ ends of reads which was performed with cutadapt, using a 75-mer oligo-A sequence as the “adapter” and a minimal overlap of 2.
Genome_build: hg19 for human data, mm10 for mouse data
Supplementary_files_format_and_content: Processed data file Polysome_htseq_readcounts.txt is a tab delimited .txt files containing raw read counts obtained from HTSEQ (0.5.4p3) at exons of genes. Also included is Polysomal_RNA_sequencing_Control_1.bw bigwig file
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| Submission date |
Apr 23, 2020 |
| Last update date |
Aug 26, 2022 |
| Contact name |
Pierre-Rene Körner |
| Organization name |
Netherlands Cancer Institute (NKI)
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| Department |
Oncogenomics
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| Street address |
Plesmanlaan 121
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| City |
Amsterdam |
| ZIP/Postal code |
1066CX |
| Country |
Netherlands |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE149204 |
Alternative cleavage and polyadenylation generates downstream uncapped RNA isoforms with translation potential |
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| Relations |
| BioSample |
SAMN14678289 |
| SRA |
SRX8163557 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4492160_Polysomal_RNA_sequencing_Control_1.bw |
11.7 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
| Processed data provided as supplementary file |
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