|
| Status |
Public on Dec 31, 2020 |
| Title |
Uninfected WT RAW Cells 1 |
| Sample type |
SRA |
| |
|
| Source name |
Modified RAW264.7 RAW Cell Line
|
| Organism |
Mus musculus |
| Characteristics |
sp-r210 isoform expression: WT RAW 264.7 cells with empty pTriex-2-Neo vector
iav infection: Uninfected
|
| Treatment protocol |
Infected cells were infected with MOI 2 of PR8 influenza A virus for 2 hours in infection media (1:1 DMEM/PBS), after which virus-containing media was removed and replaced with DMEM with 10% FBS. Infection was allowed to porceed for 22 hour for a total 24 hour infection period. Control cells had media replaced with infection media for 2 hours, after which 10% FBS containing DMEM was replaced for the remainder of the time.
|
| Growth protocol |
RAW264.7 cells were stably transfected with empty for SP-R210ct pTriex-2-Neo vectors and selected using 600ug/mL neomycin sulfate to generate WT and SP-R210L(DN) RAW264.7 cells, respectively. SP-R210KO cells were generated using CRISPR/Cas9. Guide RNA sequences targeting exons 5, 6, 12, and 14 were designed using crispr.mit.edu and gRNA oligonucleotides were ligated into the LRG hU6-sgRNA- EFS-GFP-P2A vector. RAW 264.7 macrophages were transduced with a Cas9 containing lentivirus and selected using blasticidin to generate a stable Cas9 expressing cell line. Stable Cas9 RAW264.7 cells were transfected with a pooled library of ligated gRNA vectors. Transfected GFP+ cells were isolated by flow activated cell sorting into 96-well plates to culture individual clones. SP-R210-deficient cells were identified by Western blot analysis. 1E6 cells of each of WT, SP-R210L(DN) and SP-R210KO cells were plated for each sample.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from 1,000,000 cells per sample using the RNA-Bee protocol. Briefly, plated cells were washed with PBS and 0.5 mL of RNA-Bee Isolation Reagent was added to the cells, lysed with repeated pipetting, and transferred to a microcentrifuge tube followed by the addition of 0.1 mL of chloroform was added and mixed by shaking for 15-30 seconds. Homogenate was centrifuged at 12,000xg for 15 minutes at 4oC. The aqueous phase was retained and mixed with 0.5 mL ice-cold isopropanol and placed at -20oC for 3 hours. Samples were then centrifuged at 12,000 x g for 15 minutes at 4oC and supernatant was discarded. Precipitated RNA was washed with 1 mL ice-cold 75% ethanol twice, and then allowed to air dry for 15-30 minutes at 4oC. RNA was dissolved in 25 uL RNAse, DNAse-free deionized water.
The cDNA libraries were prepared using the NEXTflex™ Illumina Rapid Directional RNA-Seq Library Prep Kit (BioO Scientific) as per the manufacturer’s instructions. Briefly, polyA RNA was purified from 200 ng of total RNA using oligo (dT) beads. The extracted mRNA fraction was subjected to fragmentation, reverse transcription, end repair, 3’– end adenylation, and adaptor ligation, followed by PCR amplification and SPRI bead purification (Beckman Coulter). The unique index sequences were incorporated in the adaptors for multiplexed high-throughput sequencing. The final product was assessed for its size distribution and concentration using BioAnalyzer High Sensitivity DNA Kit (Agilent Technologies). .
Pooled libraries were diluted and then denatured using the Illumina protocol. The denatured libraries were loaded onto a TruSeq v2 Rapid flow cell on an Illumina HiSeq 2500 and run for 50 cycles using a single-read recipe or onto a S1 flow cell on an Illumina NovaSeq 6000 and run for 2X50 cycles according to the manufacturer's instructions.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
processed data file:
WTRNA_InfvUninf.csv
|
| Data processing |
De-multiplexed and adapter-trimmed sequencing reads were aligned to the mm10 reference genome using hisat2. Abundance for each gene was obtained using featureCounts function available in Rsubread R package. The raw count data from 3 independent replicates were analyzed using DESeq2 to obtain differentially expressed genes with p-value <0.05
Genome_build: mm10
Supplementary_files_format_and_content: Procesed Deseq results providing differentially expressed genes with p-value <0.05 between cell type are in the correspondingly labeled CSV file. Deseq results comparing infected samples without filtereing are in the correspondingly labeled CSV file.
|
| |
|
| Submission date |
Dec 30, 2020 |
| Last update date |
Jan 01, 2021 |
| Contact name |
Zissis Chroneos |
| E-mail(s) |
zchroneos@pennstatehealth.psu.edu
|
| Organization name |
Penn State College of Medicine
|
| Department |
Pediatrics
|
| Street address |
500 University Drive
|
| City |
Hershey |
| State/province |
PA |
| ZIP/Postal code |
17036 |
| Country |
USA |
| |
|
| Platform ID |
GPL17021 |
| Series (2) |
| GSE164035 |
Genomic and epigenomic adaptation in SP-R210 (Myo18A) isoform-deficient macrophages [RNA-seq] |
| GSE164036 |
Genomic and epigenomic adaptation in SP-R210 (Myo18A) isoform-deficient macrophages |
|
| Relations |
| BioSample |
SAMN17184585 |
| SRA |
SRX9756308 |
