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Sample GSM7761782 Query DataSets for GSM7761782
Status Public on Oct 03, 2023
Title IP_NSP9_12h_KO_Rep2_cRIP
Sample type SRA
 
Source name SARS-CoV-2 infected A549-ACE2 SND1 knockout cells (KO)
Organism Homo sapiens
Characteristics cell line: A549-ACE2
genotype: SND1 knockout
infection: SARS-CoV-2
rip antibody: NSP9
Treatment protocol For SND1 eCLIP, N eCLIP with UV crosslinking (+XL), and N eCLIP without UV crosslinking (noXL), we grew 24 million cells per condition and infected them with SARS-CoV-2 at MOI 3 PFU/cell. At 24 hpi, culture media was removed, cells were briefly rinsed with PBS and subjected to UV-irradiation with a total dose of 0.8 J/cm2, except N noXL samples, which were not UV crosslinked. Cells were scraped in PBS, pelleted at 200x g for 5 min and lysed by adding 2x CLIP lysis buffer (100 mM Tris-HCl pH 7.4, 300 mM NaCl, 2 mM EDTA, 2% (v/v) IGEPAL CA-630, 1% sodium deoxycholate, 0.5 mM DTT, EDTA-free Protease Inhibitor Cocktail). Following a 30 min incubation at room temperature, fresh lysates were lysates were directly used for immunoprecipitation procedures or stored at -80°C.
For NSP9 cRIP experiments, we grew 24 million cells per condition and infected them with SARS-CoV-2 at MOI 3 PFU/cell. At 8 hpi and 12 hpi, culture media was removed and cells were scraped in cold PBS without UV crosslinking. Cells were pelleted by centrifugation (200 g, 8 min, 4°C) and lysed by adding 2x Co-IP buffer (100 mM Tris-HCl pH 7.4, 300 mM NaCl, 2% (v/v) IGEPAL CA-630, 1% sodium deoxycholate, 0.5 mM TCEP, EDTA-free Protease Inhibitor Cocktail). Following a 30 min incubation at room temperature, fresh lysates were used for immunoprecipitation procedures.
Growth protocol We maintained HuH-7 or A549-ACE2 cells in DMEM media (Thermo Fisher Scientific) supplemented with 10% heat-inactivated FBS (Thermo Fisher Scientific), and 100 units/ml streptomycin and 100 mg/ml penicillin. Cells were grown at 37°C and 5% CO2 atmosphere.
Extracted molecule total RNA
Extraction protocol For immunoprecipitation procedures, lysates were combined with an equal amount of nuclease free water to adjust the lysis buffer to a 1x concentration. Subsequent steps were performed as described in the eCLIP protocol, with the following modifications. Immunoprecipitates were washed two times in 1 ml CLIP lysis buffer, two times in IP wash buffer (50 mM Tris-HCl pH 7.4, 300 mM NaCl, 1 mM EDTA, 1% (v/v) NP40, 0.5% sodium deoxycholate, 0.25 mM DTT), followed by two washes in 50 mM Tris-HCl pH 7.4, 1 mM EDTA, 0.5% (v/v) NP40. All other steps were carried out as described in the eCLIP method (Nostrand, E. L. V. et al. Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP). Nat Methods 13, 508–14 (2016)).
eCLIP-seq (Nostrand, E. L. V. et al. Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP). Nat Methods 13, 508–14 (2016))
 
Library strategy RIP-Seq
Library source transcriptomic
Library selection other
Instrument model Illumina NextSeq 500
 
Description second replicate
Data processing Paired-end sequencing reads from cRIP experiments, were adapter- and quality trimmed by cutadapt (v1.18) in two runs according to the ENCODE eCLIP-seq processing pipeline v. 2.2 (Nostrand, E. L. V. et al. Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP). Nat Methods 13, 508–14 (2016)). In the first run 5' and 3' adapters were clipped from both reads using the parameters: --match-read-wildcards --times 1 -e 0.1 -O 1 --quality-cutoff 6 -m 18 -a NNNNNAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC -g CTTCCGATCTACAAGTT -g CTTCCGATCTTGGTCCT -A AACTTGTAGATCGGA -A AGGACCAAGATCGGA -A ACTTGTAGATCGGAA -A GGACCAAGATCGGAA -A CTTGTAGATCGGAAG -A GACCAAGATCGGAAG -A TTGTAGATCGGAAGA -A ACCAAGATCGGAAGA -A TGTAGATCGGAAGAG -A CCAAGATCGGAAGAG -A GTAGATCGGAAGAGC -A CAAGATCGGAAGAGC -A TAGATCGGAAGAGCG -A AAGATCGGAAGAGCG -A AGATCGGAAGAGCGT -A GATCGGAAGAGCGTC -A ATCGGAAGAGCGTCG -A TCGGAAGAGCGTCGT -A CGGAAGAGCGTCGTG -A GGAAGAGCGTCGTGT
In the second run 3' adapters of read 2 were clipped to control for double ligation events. Cutadapt clipping was performed with the parameters: --match-read-wildcards --times 1 -e 0.1 -O 5 --quality-cutoff 6 -m 18 -A AACTTGTAGATCGGA -A AGGACCAAGATCGGA -A ACTTGTAGATCGGAA -A GGACCAAGATCGGAA -A CTTGTAGATCGGAAG -A GACCAAGATCGGAAG -A TTGTAGATCGGAAGA -A ACCAAGATCGGAAGA -A TGTAGATCGGAAGAG -A CCAAGATCGGAAGAG -A GTAGATCGGAAGAGC -A CAAGATCGGAAGAGC -A TAGATCGGAAGAGCG -A AAGATCGGAAGAGCG -A AGATCGGAAGAGCGT -A GATCGGAAGAGCGTC -A ATCGGAAGAGCGTCG -A TCGGAAGAGCGTCGT -A CGGAAGAGCGTCGTG -A GGAAGAGCGTCGTGT
RNA-seq reads were aligned to the concatenated genomes of hg38 (ensembl) and SARS-CoV-2 (ensembl MN908947.3) using STAR version 2.7.10a with parameters --outFilterScoreMinOverLread 0 --outFilterMatchNminOverLread 0 --outFilterMatchNmin 0 --outFilterType Normal --alignSoftClipAtReferenceEnds No --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --outFilterMismatchNoverLmax 0.04 --scoreDelOpen -1 --alignIntronMin 20 --alignIntronMax 3000 --alignMatesGapMax 3000 --alignEndsType EndToEnd
Using the python package pyBigWig, bigWig files were generated based on the SARS-Cov-2 genome and files were separately created for positive and negative sense mapped reads. Score represents the information content of IP over SMI of positive and negative sense RNA fragments at a given genomic coordinate.
Assembly: hg38 and MN908947.3
Supplementary files format and content: bw
 
Submission date Sep 05, 2023
Last update date Oct 03, 2023
Contact name Alexander Gabel
E-mail(s) alexander.gabel@helmholtz-hiri.de
Organization name Helmholtz Institute for RNA-based Infection Research
Lab LncRNA and Infection Biology (LRIB)
Street address Josef-Schneider-Str. 2 / D15
City Würzburg
ZIP/Postal code 97080
Country Germany
 
Platform ID GPL18573
Series (1)
GSE217429 SND1 binds SARS-CoV-2 negative-sense RNA and promotes viral RNA synthesis through NSP9
Relations
BioSample SAMN37285188
SRA SRX21637697

Supplementary file Size Download File type/resource
GSM7761782_IC_NSP9_12h_KO_Rep2_neg_sense.bw 148.1 Kb (ftp)(http) BW
GSM7761782_IC_NSP9_12h_KO_Rep2_pos_sense.bw 229.8 Kb (ftp)(http) BW
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