|
| Status |
Public on Mar 27, 2023 |
| Title |
TIR-STING induced, harvested at 18 hr, replicate 2 |
| Sample type |
SRA |
| |
|
| Source name |
phage ORF library
|
| Organism |
Bacteriophage sp. |
| Characteristics |
tissue: phage ORF library
construct: pET30a encoding Flag-TIR-STING
condition: TIR-STING induced, harvested at 18 hr
|
| Growth protocol |
Plasmid DNA encoding the pooled ORF library was then prepared and used to transform BL21 (DE3) Rosetta 2 competent cells (MilliporeSigma) together with pET30a encoding Flag-TIR-STING or empty vector control. 100 ml of overnight cultures were diluted 1:20 and grown in M9 media supplemented with 0.25% CAA and 1% glycerol, and induced at A600=0.3-0.4 through the addition of 0.5mM IPTG. Samples were taken immediately prior to induction and at 18 h post-induction.
|
| Extracted molecule |
other |
| Extraction protocol |
Plasmid DNA was recovered by miniprep, and library inserts amplified by PCR using primers flanking the ORF insertion site in pET-SUMO2.
Bar-coded libraries for Illumina sequencing were generated using the sparQ DNA Library Kit (Quantabio) and 50 bp reads generated using a HiSeq3000 sequencing system.
|
| |
|
| Library strategy |
OTHER |
| Library source |
other |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 3000 |
| |
|
| Description |
Double-stranded gene fragments and pooled, single-stranded DNA oligonucleotides encoding phage ORFs with 20-25 nt flanking regions of homology to the pET-SUMO2 vector flanking the NdeI and NotI sites were purchased from IDT (eBlocks and oPools). ssDNA oligos were converted to dsDNA through isotheral extension of a primer complementary to the 3’ flanking region (sparQ HiFi PCR Master Mix, Quantabio), and products purified with a DNA spin column. Pooled ORFs were inserted into pET-SUMO2 by DNA assembly (NEBuilder HiFi) and transformed into E. coli (NEB DH5a). Colonies were scraped from plates, combined and inoculated into 10 ml liquid cultures. Plasmid DNA encoding the pooled ORF library was then prepared and used to transform BL21 (DE3) Rosetta 2 competent cells (MilliporeSigma) together with pET30a encoding Flag-TIR-STING or empty vector control. 100 ml of overnight cultures were diluted 1:20 and grown in M9 media supplemented with 0.25% CAA and 1% glycerol, and induced at A600=0.3-0.4 through the addition of 0.5mM IPTG. Samples were taken immediately prior to induction and at 18 h post-induction. Plasmid DNA was recovered by miniprep, and library inserts amplified by PCR using primers flanking the ORF insertion site in pET-SUMO2. Bar-coded libraries for Illumina sequencing were generated using the sparQ DNA Library Kit (Quantabio) and 50 bp reads generated using a HiSeq3000 sequencing system. Raw reads were trimmed to remove adapters and low-quality reads using Trim Galore (v0.3.5). Bowtie 1.3.1 (Langmead et al. 2009) was used to align reads and Cufflinks (v2.2.1) (Trapnell et al. 2010) was used to estimate read abundances and test for differential expression.
|
| Data processing |
Raw reads were trimmed to remove adapters and low-quality reads using Trim Galore (v0.3.5).
Bowtie 1.3.1 (Langmead et al. 2009) was used to align reads and Cufflinks (v2.2.1) (Trapnell et al. 2010) was used to estimate read abundances and test for differential expression.
Assembly: Reads aligned to attached fasta file "amplicon_sequence.fa" - a custom library encoding bacteriophage genes.
|
| |
|
| Submission date |
Jan 03, 2023 |
| Last update date |
Mar 27, 2023 |
| Contact name |
Douglas Edmund Feldman |
| E-mail(s) |
defeldma@usc.edu
|
| Organization name |
USC Keck School of Medicine
|
| Department |
Pathology
|
| Lab |
HMR 212
|
| Street address |
2011 Zonal Avenue
|
| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90033 |
| Country |
USA |
| |
|
| Platform ID |
GPL32997 |
| Series (1) |
| GSE222071 |
Bacteriophage antidefense genes that neutralize TIR and STING immune responses |
|
| Relations |
| SRA |
SRX18913238 |
| BioSample |
SAMN32542991 |
