NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM6718732 Query DataSets for GSM6718732
Status Public on Oct 03, 2023
Title SMI_NSP9_8h_CTRL_Rep1_cRIP
Sample type SRA
 
Source name SARS-CoV-2 infected A549-ACE2 control cells (CTRL)
Organism Homo sapiens
Characteristics cell line: A549-ACE2
genotype: control (CTRL)
infection: SARS-CoV-2
rip antibody: NA
Treatment protocol For SND1 eCLIP experiments, 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. 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
 
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 Nov 07, 2022
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 SAMN31636193
SRA SRX18192546

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data not provided for this record

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap