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Sample GSM2098861

Query DataSets for GSM2098861

Status

Public on Aug 26, 2016

Title

11_CP_30

Sample type

SRA

Source name

A549_capsular mutant, more adherent_30m

Organisms

Homo sapiens; Streptococcus pneumoniae D39

Characteristics

cell line: A549
cell type: human epithelial cell line
timepoints (mpi): 30
pneumococcal strain: capsular mutant, more adherent
molecule subtype: total epithelial and pneumococcal RNA

Treatment protocol

Various co-incubation time between human epithelial cell line A549 and S. pneumoniae D39, including 0, 30, 60, 120 and 240 minutes after co-incubation/infection.

Growth protocol

Human epithelial cell line A549 cells were grown in DMEM/F12 medium supplemented with 10%FBS in 37C, humidified 5% CO2. S. pneumoniae D39 were grown in C+Y, 37C. Both species were co-incubated in an infection model, in RPMI 1640 without phenol red supplemented by 1%FBS, 37C 5% CO2.

Extracted molecule

total RNA

Extraction protocol

To harvest the total RNA from host and pathogen at once, we treated cellular mixture with concentrated solution of ammonium sulfate to prevent any protein-dependent RNA degradation. Each ml of the ammonium sulfate solution (pH 5.2) contains 0.7 g (NH₄)₂SO₄, 20 mM EDTA and 25 mM Na-citrate. Ammonium sulfate solution is added directly to the medium with the ratio 3:1. The suspension was vigorously pipetted to ensure the complete mixing of ammonium sulfate and infection medium. Adherent host cells were scrapped and incubated further (room temperature, 5 min). The suspension is collected and centrifuged at full speed (20 min, 4°C). Supernatant was removed and cell pellet was snap-frozen with liquid nitrogen. The cell mixture contains host cells, adherent bacteria and non-adherent bacteria. With the aim of disrupting pneumococcal cells, a bead beating procedure was exercised. In a 1.5 ml screw cap tube, a PCR tube full of sterile, RNase free glass beads were added together with 50 μl 10% SDS and 500 μl phenol-chloroform. In the meantime, frozen cell pellet was resuspended in TE solution (10 mM Tris-HCl, 1 mM Na₂EDTA, pH 8.0, DEPC, diethylpyrocarbonate-treated solution). The cell suspension was added into the screw-capped tube and beat for 3 times 45s. Tubes were immediately placed in ice box and centrifuged at full speed, 4°C to separate organic and water phases. Water phase was pipetted out and back extraction were performed to the organic phase to optimize RNA yield. The subsequent part of the RNA isolation were performed based on High Pure RNA Isolation Kit (Roche) with column. Water phase from phenol-chloroform extraction were mixed well with binding buffer and pipetted into the upper chamber of column and centrifuged. DNase mix were then added onto the filter and incubated at room temperature for 30 min to digest total genomic DNA. Total RNA were eluted according to the manufacturer protocol. Total RNA samples were treated with a 1:1 mixture of Human/Mouse/Rat and Gram positive bacteria capture probes (Ribo-Zero rRNA Removal Kits, Illumina, US) to simultaneously deplete host and pathogen rRNA. cDNA library preparation was prepared with TruSeq® Stranded Total RNA Sample Preparation Kit (Illumina, US) according to the manufacturer protocol.
RNA libraries were prepared for sequencing using standard Illumina protocols

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

the sample contains total RNA from the human cell line and the bacteria S. pneumoniae

Data processing

Adapter sequences were removed, and raw reads were trimmed with sliding window every 5 nucleotides, the average value must be more than 20, final trimmed length must be more than 66.7% original length (more than 50 nucleotides out of original 75 nucleotides).
Reads were aligned to a chimeric genome, containing a concatenated S. pneumoniae D39 genome to Homo sapiens genome.
Aligned reads were counted at the gene level.
Genome_build: Streptococcus pneumoniae D39 build NC_008533 and Homo sapiens GRCh.38
Supplementary_files_format_and_content: Normalized raw counts by DESeq2 in MS Excel 2010

Submission date

Mar 24, 2016

Last update date

May 15, 2019

Contact name

Rieza Aprianto

E-mail(s)

R.Aprianto@rug.nl

Phone

+31503632409

Organization name

University of Groningen