|
| Status |
Public on Nov 19, 2019 |
| Title |
12hr_infected.E5.batch3 |
| Sample type |
SRA |
| |
|
| Source name |
Epithelial cells (HEp2)
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: epithelial (HEp2)
condition: infected
hours post infection: 12
sequencing batch: 3
chlamydial species: Chlamydia trachomatis - Serovar E
|
| Treatment protocol |
Chlamydia trachomatis serovar E infections of HEp-2 cells and matched mock infections proceeded to 3, 6, and 12 hours post-infection before monolayer dissociation with trypsin.
|
| Growth protocol |
HEp-2 cells (American Type Culture Collection, ATCC No. CCL-23) were grown as monolayers in 6 x 100mm TC dishes until 90% confluent. Fresh monolayers were infected with Chlamydia trachomatis serovar E in 3.5 mL SPG buffer for an MOI ~ 1, using centrifugation to synchronize infections. Infections and subsequent culture were performed in the absence of cycloheximide or DEAE dextran. A matching number of HEp-2 monolayers were also mock-infected using uninfected cell lysates. Each treatment was incubated at 25°C for 2h and subsequently washed twice with SPG to remove dead or non-viable EBs. 10 mL fresh medium (DMEM + 10% FBS, 25μg/ml gentamycin, 1.25μg/ml Fungizone) was added and cell monolayers incubated at 37°C with 5% CO2.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Single cell suspensions of time-matched mock-infected and Chlamydia trachomatis E-infected cells at 3, 6, and 12 hours post-infection were processed using the Fluidigm C1 scRNA-seq protocol according to the manufacturers instructions.
scRNA-Seq libraries were constructed using the standard protocol described for the Fluidigm C1 scRNA-seq.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
Base calls were performed using the bulit in Illumina basecaller and demultiplexed using DeML
Sequenced reads were trimmed and quality checked using Trim Galore (v.0.4.3), FastQC (v.0.11.5) and FastQ-Screen (v.0.11.1)
Trimmed reads were aligned to the human genome (GRCh 38.87) using STAR (v.2.5.1a) keeping paired and unpaired aligned reads, genomic features were counted with FeatureCounts (v.1.5.0-p3)
Genome_build: Genome Reference Consortium Human Build 38 patch release 87 (GRCh38.p87)
Supplementary_files_format_and_content: Count matricies generated from each of the raw BAM files using FeatureCounts
|
| |
|
| Submission date |
Jun 11, 2019 |
| Last update date |
Nov 23, 2019 |
| Contact name |
Regan Hayward |
| Organization name |
Helmholtz Institute for RNA-based Infection Research
|
| Street address |
Josef-Schneider-Straße 2 / D15
|
| City |
Wurzburg |
| ZIP/Postal code |
97080 |
| Country |
Germany |
| |
|
| Platform ID |
GPL20301 |
| Series (1) |
| GSE132525 |
Early transcriptional landscapes of Chlamydia trachomatis-infected epithelial cells at single cell resolution |
|
| Relations |
| BioSample |
SAMN12017201 |
| SRA |
SRX6037556 |
