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Sample GSM5268697 Query DataSets for GSM5268697
Status Public on Mar 09, 2022
Title NSP13 rep2 IP
Sample type SRA
Source name BEAS-2B expressing NSP13
Organism Homo sapiens
Characteristics antibody manufacturer: Sigma-Aldrich
antibody catalog #: Monoclonal ANTI-FLAG® M2 antibody produced in mouse, Sigma Aldrich, F3165
antibody: 3XFLAG tag
adapter: InvRNA2
Treatment protocol Cells were UV cross-linked with 400 mJ/cm^2 and processed through the eCLIP protocol as previously described (van Nostrand et al, 2016). Anti-Strep tag or Anti-FLAG tag antibody was employed for immunoprecipitation.
Vero E6 cells were infected with SARS-CoV-2 at MOI of 0.02 for 48 hours before UV crosslinking with 400 mJ/cm^2. Anti-N, Anti-NSP8 and Anti-NSP12 antibodies were used for immunoprecipitation.
Growth protocol BEAS-2B cells were cultured on Matrigel (Corning) coated plates and maintained in the PneumaCult-Ex Plus Medium (Stem Cell Technologies), supplemented with 33 µg/ml hydrocortisone (Stem Cell Technologies). Growth media was replaced every two days, and the cells were passaged every four days. Vero E6 cells were cultured in DMEM (ThermoFisher) supplemented with 10% FBS (ThermoFisher) and passaged every three days. All cell cultures were incubated at 37ºC and 5% CO2.
Vero E6 cells were cultured in DMEM supplemented with 10% FBS.
Extracted molecule total RNA
Extraction protocol Biological replicates (according to ENCODE requirements) of 20 million cells were treated with 400mJ of UV using the Stratalinker 2400, harvested by scraping in ice cold PBS and pellets flash frozen in liquid nitrogen and stored in -80 degrees until ready to IP with antibody bound Dynabeads Subsequent protocol exactly as detailed in Van Nostrand et al 2016. Sequencing was performed on Illumina 2000 Hi Seq paired end reads and data processed through Dr. Yeo's eCLIP pipeline.
Libraries were constructed as per the eCLIP protocol using Illumina 50x forward and 70x reverse primers.
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina HiSeq 2000
Data processing Sequenced reads were reformatted to include randomers in read headers with umi_tools (1.0.0). Args: --random-seed 1 --bc-pattern NNNNNNNNNN
Reads were then trimmed with cutadapt (1.14). Args: --match-read-wildcards -O 1 --times 1 -e 0.1 --quality-cutoff 6 -m 18 -a InvRNA*.fasta (fasta sequences can be found at:
Reads were then trimmed once more with cutadapt (1.14) to remove double-ligation events. Args: --match-read-wildcards -O 5 --times 1 -e 0.1 --quality-cutoff 6 -m 18 -a InvRNA*.fasta (fasta sequences can be found at:
Trimmed reads were then mapped with STAR (2.4.0i) against a repeat element database (RepBase 18.05). Args: --runThreadN 16 \ --genomeDir human_repbase \ --readFilesIn path/to/read1 \ --outFileNamePrefix out_prefix \ --outReadsUnmapped Fastx \ --outSAMtype BAM Unsorted \ --outSAMattributes All \ --outSAMunmapped Within \ --outSAMattrRGline ID:foo \ --outFilterType BySJout \ --outFilterMultimapNmax 30 \ --outFilterMultimapScoreRange 1 \ --outFilterScoreMin 10 \ --alignEndsType EndToEnd
Unmapped reads filtered of repeat elements were then mapped with STAR (2.4.0i) against a human genome (hg19/ChlSab2). Args: --runThreadN 16 \ --genomeDir genomedir \ --readFilesIn /path/to/read1 \ --outFileNamePrefix out_prefix \ --outReadsUnmapped Fastx \ --outSAMtype BAM Unsorted \ --outSAMattributes All \ --outSAMunmapped Within \ --outSAMattrRGline ID:foo \ --outFilterType BySJout \ --outFilterMultimapNmax 1 \ --outFilterMultimapScoreRange 1 \ --outFilterScoreMin 10 \ --alignEndsType EndToEnd
Aligned reads were sorted with samtools (1.6)
Sorted reads were collapsed with umi_tools (1.0.0). Args: --random-seed 1 --method unique
BAM files were used to identify peak clusters with Clipper (1.2.2). Args: --species (hg19/ChlSab2_Sars) --bam path/to/input.bam --timeout 3600000 --maxgenes 1000000 --save-pickle --outfile path/to/output.bam
Peak clusters were normalized using BAM files for IP against BAM files for INPUT with (, included in eclip 0.1.5+.
Overlapping normalized peak regions were merged with, included within eclip-0.1.5+
Normalized peak (compressed.bed) files were ranked by entropy score ( included within the merge_peaks pipeline) and used as inputs to IDR (2.0.2) to determine reproducible peaks.
Reproducible peaks were filtered for those ≥20 bases in length, and not overlapping with WT negative control samples.
Genome_build: hg19
Genome_build: ChlSab2
Genome_build: Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1, complete genome (MN908947.3)
Submission date Apr 28, 2021
Last update date Mar 09, 2022
Contact name Gene Yeo
Organization name UCSD
Street address 2880 Torrey Pines Scenic Dr. Room 3805/Yeo Lab
City La Jolla
State/province CA
ZIP/Postal code 92037
Country USA
Platform ID GPL11154
Series (2)
GSE173498 Discovery and functional interrogation of the virus and host RNA interactome of SARS-CoV-2 proteins [eCLIP]
GSE173508 Discovery and functional interrogation of the virus and host RNA interactome of SARS-CoV-2 proteins
BioSample SAMN18906584
SRA SRX10697865

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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