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| Status |
Public on May 11, 2024 |
| Title |
18hr MVA infected RNA-seq Bio rep 1 |
| Sample type |
SRA |
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| Source name |
Kidney derived cell line
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| Organism |
Chlorocebus sabaeus |
| Characteristics |
tissue: Kidney derived cell line
cell line: Vero CCL-81
cell type: Kidney derived cell line
treatment: 18hr MVA infected
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| Treatment protocol |
Cryopreserved Vero cells were thawed at passage #8 and propagated for 2 passages prior toseeding for viral infection. At the time of seeding, a cell suspension was made in pre-warmedcomplete medium and digitally counted using the Scepter 3.0 Handheld Cell Counter and 60 μmCounter Sensors (Millipore, Cat # PHCC360KIT). Cells were seeded for viral infection atpassage #11 into replicate tissue culture flasks and multi-well plates using 5x10^6 cells per T175flask or 5x10^5 cells per well in 12-well plates. Seeded flasks and plates were incubated for 24 hrsat 37°C, yielding ~1x10^7 cells per flask and ~1x10^6 cells per well at the time of infection. Modified Vaccinia Ankara virus, strain VACV-MVA, was obtained from the ATCC (Cat # VR-1508). VACV-MVA viral stock was diluted in infection medium consisting of DMEM31supplemented with 2% (v/v) FBS. Vero cells were infected at 0 hrs using 0.4 MOI in a minimalvolume of inoculum (3 mL per flask) for 1 hr at 37°C; 17 mL infection medium was added priorto incubation for 12-24 hrs at 37°C. In parallel to the flasks, 12-well plates for ICC staining andcell imaging were also infected in parallel to the flasks at 0 hr using 0.4 MOI in 300 μL per well;then 700 μL infection medium was added prior to incubation for 12-24 hrs at 37°C.
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| Growth protocol |
Cryopreserved Vero cells, derived from the kidney tissue of a normal adult African greenmonkey, were sourced from the ATCC (Cat # CCL-81). Cells were thawed, passaged, expanded,and seeded for viral infection in complete medium consisting of Dulbecco’s Modified Eagle’sMedium (DMEM) containing 4 mM L-Glutamine (ATCC, Cat # 30-2002), supplemented with10% (v/v) Fetal Bovine Serum (FBS, ATCC, Cat # 30-2020) and filter-sterilized beforeuse. Vero cells were maintained at 37°C with 5% CO2 and 95% relative humidity. Vero cellswere cryopreserved at passage #7 from the original source vial using freeze medium consistingof DMEM supplemented with 10% (v/v) FBS and 5% (v/v) dimethylsulfoxide (DMSO, ATCC,Cat # 4-X).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were harvested in two biological replicates (A and B) per condition (mock-infected andMVA-infected) at each timepoint (12 hrs, 18 hrs, and 24 hrs post-infection). At each timepoint,cells were washed using 1X phosphate-buffered saline (PBS, Gibco, Cat # 10010049), detachedfor 15 min at 37°C using 1X TrypLE Express Enzyme (Gibco, Cat # 12604021), andresuspended in pre-warmed complete medium before cell counting with the Scepter 3.0Handheld Cell Counter and 60 μm Counter Sensors. At each timepoint, mock-infected andMVA-infected cell suspensions were divided and processed for Hi-C, ATAC-seq and RNA-seqtechniques
For RNA-seq, 2x10^5 cells were cryopreserved in duplicate 1 mL aliquots using freeze mediumon the day of cell harvest. Aliquots were subsequently thawed in a 37°C water-bath into ice-coldcomplete medium, then pelleted by centrifugation, washed in ice-cold HBSS, and resuspended in350 μL ice-cold RULT Buffer (Qiagen, Cat # 73934) containing 1% beta-mercaptoethanol(Sigma, Cat #63689). RNA was extracted from these cell lysates using the RNeasy UCP MicroKit protocol and reagents (Qiagen, Cat #73934). DNA was digested via on-column DNase Itreatment for 15 min at room temperature. RNA extracts were eluted in 20 μL RNase-free water.The concentration of total RNA was determined using the Qubit RNA HS Assay Kit(ThermoFisher Scientific, Cat # Q32855) and quality was measured using the Bioanalyzer RNA6000 Assay (Agilent, Cat. # 5067-1511). Ribosomal RNA was depleted from 500 ng total RNAusing the Illumina Ribo-Zero Plus rRNA Depletion Kit (Illumina, Cat. # 20037135). RNA wasconverted to cDNA, and adapters and indexes were added onto the ends of the fragments togenerate strand specific Illumina libraries. Libraries were eluted in DNA Elution Buffer (ZymoResearch, Cat #D3004-4-10). The concentration and average size of the libraries was verifiedusing the TapeStation DNA 5000 Assay Reagents and Screen Tapes (Agilent, Cat. # 5067-5589and 5067-5588). Libraries were normalized and pooled based on the TapeStation concentrations.An accurate library quantification of the pool was determined using the Illumina/Universal Kit(KAPA Biosystems, KK4824). The pool was sequenced on an entire lane of a NextSeq 500 HOflow cell to generate paired-end 151 bp reads using the NextSeq 500 High Output Kit v2.5 (300cycles) (Illumina, Cat # 20024908).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
2501_134-UDP0077-_TCTGGTATCC-CGTTGCTTAC__S23_R1_PAIREDAligned.out.sam.counts
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| Data processing |
The Chlorocebus sabaeus reference genome was retrieved from NCBI (GCF_000409795.2) andappended with the vaccinia virus (VACV) genome, retrieved from GenBank (U94848.1).Genome annotations for C. sabaeus were downloaded from NCBI and the GenBank flat fileannotations for VACV were retrieved using the bio package command (bio fetch U94848.1 --format gff) and converted to gtf format using the AGAT package function(agat_convert_sp_gff2gtf.pl). The C. sabaeus annotation was appended with the VACVannotation for subsequent analysis. Raw read quality control was performed using FastQC andSeqMonk to verify library quality and degree of PCR duplication. All metrics were normal. Asplice junction aware reference index was generated for the combined genome and annotationfiles using STAR with the arguments (--runMode genomeGenerate --sjdbOverhang 149). Prealignmentread trimming was not performed in favor of soft-clipping/quality filtering by theSTAR aligner. Reads were aligned to the combined genome using STAR. Duplicate reads weremarked but retained in downstream analysis per DESeq2 usage guidelines. Sense strand readsaligning to gene features were counted using htseq-count with the arguments (--stranded=reverse). Antisense strand reads aligning to gene features were counted using htseqcountwith the arguments (--stranded=yes). Differential expression analysis was performed usingthe DESeq2 matrix design (~time + treatment). For visualization, replicate alignment files weremerged using samtools. bigWigs were then generated using the bamCoverage function from thedeepTools package with the arguments (--effectiveGenomeSize 2836634000 --normalizeUsingBPM –filterRNAstrand {forward/reverse/NULL} --binSize 1).
Assembly: The Chlorocebus sabaeus reference genome (GCF_000409795.2) appended with the vaccinia virus (VACV) genome, retrieved from GenBank (U94848.1)
Supplementary files format and content: Strand specific read pileup in bigwig format. Including vaccinia virus genome.
Supplementary files format and content: Processed data given as supplementary files include DESEQ2 result files for various comparions. In file named UTR indicates Mock infected control and V indicates MVA infected sample. Futher, time point is mentioned in the file name to indicate the timepoints compared in each file. Comparison is made by considering control as the denominator or ealier timepoint as the denominator.
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| Submission date |
Nov 16, 2023 |
| Last update date |
May 11, 2024 |
| Contact name |
Christina Rene Steadman |
| Organization name |
Los Alamos National Laboratory
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| Department |
Earth & Environmental Sciences Division
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| Lab |
Epigenetics Lab
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| Street address |
Bikini Atoll Rd
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| City |
Los Alamos |
| State/province |
New Mexico |
| ZIP/Postal code |
87545 |
| Country |
USA |
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| Platform ID |
GPL28836 |
| Series (2) |
| GSE248050 |
Vaccinia virus infection induces concurrent alterations in host chromatin architecture, accessibility, and gene expression [RNA-seq] |
| GSE248052 |
Vaccinia virus infection induces concurrent alterations in host chromatin architecture, accessibility, and gene expression |
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| Relations |
| BioSample |
SAMN38165928 |
| SRA |
SRX22432927 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7905267_2501_134-UDP0077-_TCTGGTATCC-CGTTGCTTAC_S23MINUS.bw |
37.7 Mb |
(ftp)(http) |
BW |
| GSM7905267_2501_134-UDP0077-_TCTGGTATCC-CGTTGCTTAC_S23PLUS.bw |
39.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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