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Status |
Public on Apr 10, 2022 |
Title |
24_del_noruxo_A |
Sample type |
SRA |
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Source name |
Human + Virus
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Organisms |
Homo sapiens; Severe acute respiratory syndrome coronavirus 2 |
Characteristics |
cell line: Calu3 infection: SARS-CoV-2 hours post infection: 24
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Treatment protocol |
For Ribo-seq libraries, all samples were treated with 100μg/mL CHX for 1 minute. Cells were placed on ice immediately after treatment, washed twice with PBS containing 100μg/mL CHX.
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Extracted molecule |
total RNA |
Extraction protocol |
For RNA-seq, cells were harvested with Tri-Reagent (Sigma-Aldrich), total RNA was extracted, and poly-A selection was performed using Dynabeads mRNA DIRECT Purification Kit (Invitrogen). For ribosome profiling, medium was aspirated from dishes, which were immediately placed on ice and rinsed with 10 ml ice-cold PBS supplemented with drugs used in pretreatment of the cells. PBS was aspirated and 800 μl ice-cold lysis buffer (20 mM Tris, pH 7.4, 250 mM NaCl, 15 mM MgCl2, 1 mM dithiothreito, 0.5% Triton X-100 and 24 U / ml Turbo DNase (Ambion, AM2239), along with any drugs used for sample treatment) was dripped onto dishes. Cells were scraped and the lysate was removed and incubated 10 min on ice. The lysate was then clarified by centrifugation for 10 min at 20,000 × g, 4°C and ∼1.1 ml supernatant was recovered. RNA-seq: mRNA sample was subjected to DNaseI treatment and 3’ dephosphorylation using FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific) and T4 PNK (NEB) followed by 3’ adaptor ligation using T4 ligase (NEB). The ligated products used for reverse transcription with SSIII (Invitrogen) for first strand cDNA synthesis. The cDNA products were 3’ ligated with a second adaptor using T4 ligase and amplified for 8 cycles in a PCR for final library products of 200-300bp.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina MiSeq |
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Description |
Ruxolitinib_cds_reads.csv.gz
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Data processing |
The reads of the ribo-seq libraries were trimmed to remove the linker CTGTAGGCACCATCAAT by using cutadapt. For the Shishkin protocol libraries the linker was AGATCGGAAGAGCACACGT. Reads aligned to hg19 based rRNA and tRNA reference were removed. Then reads were aligned to combined reference of the host and the virus. Reads that failed to align to genome, were aligned to the host transcriptome and to the viral breaking points according to strong break point at Kim et al., plus few breakpoints from our STAR runs. The alignments above were made with Bowtie1. In the ribo-seq libraries the p-site was calculated as an offset (adjusted to the read length) from the read start site. The STAR alignment was made with the flags recommended by Kim et al. to get all the breakpoints. Assembly: Host: Human hg19 (GRCh37); Chlorocebus sabaeus genome and annotation ENSEMBL release 99; Mesocricetus auratus genome and annotation ENSEMBL release 104; Virus Genbank NC_045512.2. Supplementary files format and content: *.csv: Comma-separated files of the cytosolic to nuclear ratio of transcripts, half-life of transcripts, and the read counts of all transcripts in each experiment (either for coding RNAs or for all transcripts).
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Submission date |
Apr 07, 2022 |
Last update date |
Apr 10, 2022 |
Contact name |
Noam Stern-Ginossar |
E-mail(s) |
noam.stern-ginossar@weizmann.ac.il
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Organization name |
Weizmann Institute
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Department |
Molecular Genetics
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Lab |
Noam Stern-Ginossar
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Street address |
234 herzel
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City |
Rehovot |
ZIP/Postal code |
7610001 |
Country |
Israel |
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Platform ID |
GPL30234 |
Series (1) |
GSE200422 |
Parsing the role of NSP1 in SARS-CoV-2 infection |
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Relations |
BioSample |
SAMN27404838 |
SRA |
SRX14777740 |
Supplementary data files not provided |
SRA Run Selector![Help](/coreweb/images/long_help4.gif) |
Raw data are available in SRA |
Processed data are available on Series record |
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