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Sample GSM6033207

Query DataSets for GSM6033207

Status

Public on Apr 10, 2022

Title

RP_FP_WT7_B

Sample type

SRA

Source name

Human + Virus

Organisms

Homo sapiens; Severe acute respiratory syndrome coronavirus 2

Characteristics

cell line: Calu3
infection: SARS-CoV-2
hours post infection: 7

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.

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.

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina MiSeq

Description

RibosomeProfiling_FP_cds_reads.csv.gz

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).
Library strategy: Ribo-seq

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

Organization name

Weizmann Institute

Department

Molecular Genetics