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Sample GSM6914212 Query DataSets for GSM6914212
Status Public on Mar 27, 2023
Title TIR-STING induced, harvested at 18 hr, replicate 1
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 SRX18913237
BioSample SAMN32542992

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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