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Sample GSM7039459

Query DataSets for GSM7039459

Status

Public on Feb 16, 2023

Title

KV_H4_20200130

Sample type

SRA

Source name

Deep sequencing of cDNA from Streptococcus pneumoniae EF3030 bacteria and normalized human broncial epithelial cells (nHBEC), alone, together, and after coincubation with Influenza A virus pH1N1.

Organism

Homo sapiens

Characteristics

treatment condition: Co-culture
human cell type: nHBEC
incubation time: 78H

Treatment protocol

Following culture of differentiated human bronchial epithelial cells on transwells, cells were infected with pH1N1 or mock infected (no pH1N1 controls) for 72 h (~50% of cells infected with IAV). pH1N1 infected cells were then exposed to 1000 CFUs of Streptococcus pneumoniae strain EF3030 for 6 h. For pneumococcal EF3030 control samples, ~50,000 CFUs of EF3030 were cultured in ALI media for 6 hours. 1ml of RNA protect was then added to all transwells and then stored in 5ml of RNAprotect Bacteria Reagent in 50 ml falcon tubes at -80C.

Growth protocol

Primary human bronchial epithelial cells were obtained from human lung tissue through the tissue donation program by the International Institute for the Advancement of Medicine . Cells were harvested from the human tracheobronchial tissues and plated onto collagen-coated 100-mm plates (Biocoat; Becton Dickinson), in BEGM medium to ~80% confluency, followed by passaging onto 6.5-mm Transwell inserts (0.4-μm pore size) with FNC coating (0407, AthenaES) and differentiated at an Air-Liquid Interface (ALI) using Lonza tissue culture reagents. Differentiation was further enhanced by adding 10 nM recombinant human neuregulin-1α/epidermal growth factor-like domain of heregulin-α (296-HR-050, R&D Systems) to the basolateral ALI media . Cells were incubated for approximately two weeks until appearance of ciliated cells and mucus production was seen.

Extracted molecule

total RNA

Extraction protocol

On the day of RNA isolation, transwell samples were thawed and the membranes cut out using a scalpel. RNAprotect and isolated membranes were centrifuged at 10,000 rpm, to pellet the membrane and any dislodged cells, and the supernatant discarded. Pellets were then incubated in 100 μL of lysis buffer (10 μL of mutanolysin, 20 μL of proteinase K, 30 μL of lysozyme, 40 μL of TE buffer) for 10 minutes. Followed by mechanical disruption in 600 μL RLT buffer (RNeasy Mini Kit, Qiagen) containing 1% ß-mercaptoethanol, using a motorized pestle for 30 seconds. RNA was then captured on the RNeasy Mini Kit columns with DNase treatment on column (Qiagen protocol).
Strand specific RNA libraries were prepared for sequencing using standard Illumina protocols

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

Total RNAs extracted from PMNs from this donor D31 were of lower quality (lower RNA integrity [RIN] values), affecting overall RNA-seq data quality

Data processing

Streptococcus pnemoniae : Sequence reads were trimmed for adaptor sequence and mapped to the EF3030 using bowtie v1.0 with parameters -mode=v --num_mismatches=2 --file-type=fastq --seedlen=28 --minins=0 --maxins=600 --library-type=fr --args='--sam' --v --gzip
Homo sapiens: Sequenced reads were trimmedfor adaptor sequence and mapped to the 2018 GRCh38 Human genome assembly using HISAT v2.0 with parameters -mismatch-penalties=6,2 --softclip-penalties=2,1 --read-gap-penalties=5,3 --ref-gap-penalties=5,3 --min-intronlen=20 --max-intronlen=500000 --score-min=L,0,-0.2 --pen-cansplice=0 --num-threads=1 --pen-noncansplice=12 --pen-canintronlen=G,-8,1 --pen-noncanintronlen=G,-8,1 --rna-strandness=RF --dta-cufflinks=1 --num-alignments=5 --minins=0 --maxins=600 --no-unal=1 --args='' --v
Streptococcus pneumoniae: Transcripts Per Million values for gene features were calculated as described in D'Mello et al. Proceedings of the National Academy of Sciences of the United States of America vol. 117,52 (2020): 33507-33518. doi:10.1073/pnas.2010428117
Homo sapiens: Transcripts Per Million values for exon features were calculated as described in D'Mello et al. Proceedings of the National Academy of Sciences of the United States of America vol. 117,52 (2020): 33507-33518. doi:10.1073/pnas.2010428117
Assembly: Streptococcus pnemoniae:EF3030 accession NZ_CP035897.1 ; Human: 2018 GRCh38 Human genome assembly GCA_000001405.15

Submission date

Feb 11, 2023

Last update date

Feb 16, 2023

Contact name

Suvarna Nadendla

Organization name

University of Maryland School of Medicine

Department

Institute for Genome Sciences

Street address

670 W.Baltimore Street