|
| 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.
eCLIP
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
eCLIP6.NSP13-2-IP.umi.r1.fq.genome-mappedSoSo.rmDupSo.norm.neg.bw
eCLIP6.NSP13-2-IP.umi.r1.fq.genome-mappedSoSo.rmDupSo.norm.pos.bw
eCLIP6.NSP13-2-IP.umi.r1.fq.genome-mappedSoSo.rmDupSo.peakClusters.normed.compressed.bed
nsp13-1.vs.nsp13-2.bed
eCLIP6.nsp13.bed.annotated_proxdist_miRlncRNA.WTfiltered
|
| 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: https://github.com/YeoLab/eclip/tree/master/example/inputs/)
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: https://github.com/YeoLab/eclip/tree/master/example/inputs/)
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 peaksnormalize.pl (overlap_peakfi_with_bam_PE.pl), included in eclip 0.1.5+.
Overlapping normalized peak regions were merged with compress_l2foldenrpeakfi_for_replicate_overlapping_bedformat.pl, included within eclip-0.1.5+
Normalized peak (compressed.bed) files were ranked by entropy score (make_informationcontent_from_peaks.pl 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 |
| E-mail(s) |
geneyeo@ucsd.edu
|
| 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 |
|
| Relations |
| BioSample |
SAMN18906584 |
| SRA |
SRX10697865 |
