|
| Status |
Public on Nov 02, 2018 |
| Title |
Blood_Platelets_Vumc-bact-contrPLT-1 |
| Sample type |
SRA |
| |
|
| Source name |
Thrombocytes
|
| Organism |
Homo sapiens |
| Characteristics |
disease status: Healthy
cell type: Platelets
condition: Control
individual id: 1
|
| Treatment protocol |
Two different strains of E. coli bacteria were used: the non-pathogenic E. coli K12 C600 and the uropathogenic E. coli O18:K1. The bacteria were grown in lysogeny broth at 37°C and 160 rpm until they reached an OD600 nm of 0.5-0.9. The cells were pelleted by centrifugation at 18 000 x g for 10 minutes and washed with SSP+ buffer. After washing, the platelet pellet was resuspended in SSP+ and used for in vitro exposure to the bacteria on a 1:1 platelet-bacteria ratio. Platelet-bacteria samples were incubated on a see-saw shaker (10 rpm) for 3 hours.
|
| Growth protocol |
Washed platelets were resuspended in SPP+ buffer and kept on a see-saw shaker for one hour before starting the experiments.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Whole blood was collected in citrate-coated BD Vacutainer tubes. Platelet rich plasma (PRP) was prepared using centrifugation at 150 x g for 15 minutes. PRP was applied to an Optiprep density gradient and spun at 350 x g for 15 minutes to decrease the leukocyte number of the suspension. After harvesting the platelet layer, cells were washed with HEPES-Tyrode buffer (10 mM HEPES, 137 mM NaCl, 2.8 mM KCl, 1 mM MgCl2,12 mM NaHCO3, 0.4 mM Na2HPO4, 5.5 mM glucose, and 0.35% bovine serum albumin [BSA]). Centrifugation was performed without break, and before every centrifugation step 400 nM prostaglandin I2 (PGI2) was added to prevent platelet activation. Incubation of washed platelets with bacteria was followed by centrifugation of the samples at 500 x g for 10 minutes. The pellets were used for RNA isolation with a standard phenol-chloroform procedure. Briefly, centrifuged cells were resuspended in 500 µl Trizol reagent, and added to Phase lock tubes together with chloroform. The RNA pellet was air-dried and resuspended in 40 µl RNase-free water. RNA quality and quantity were measured using Bioanalyzer 2100 with RNA 6000 Picochip.
100-500 pg of platelet total RNA diluted in nuclease-free H2O was subjected to cDNA synthesis and amplification using the SMARTer Ultra Low RNA Kit for Illumina Sequencing v3 (Clontech, cat. nr. 634853) according to the manufacturer's protocol. Conversion and efficient amplification of cDNA was quality-controlled using the Bioanalyzer 2100 with DNA High Sensitivity chip (Agilent). Samples with detectable fragments in the 300-7500 bp region were selected for further processing and Covaris shearing by sonication (Covaris Inc). Sample preparation for Illumina sequencing was performed using the TruSeq DNA Sample Preparation Kit (Illumina, cat nr. FC-121-2001) or TruSeq Nano DNA Sample Preparation Kit (Illumina, cat nr. FC-121-4001). Sample quality and quantity were measured using the DNA 7500 chip or DNA High Sensitivity chip (Agilent). High-quality samples with product sizes between 300-500 bp were pooled in equimolar concentrations (12-19 samples per HiSeq lane) and submitted for 100 bp Single Read sequencing on the HiSeq 2500 platform (Illumina).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Vumc-bact-contrPLT-1
Vumc-bact-contrPLT-TR2023
processed data file: allSamples_readCounts_ISreads.txt
|
| Data processing |
Single-end 100 bp reads were subjected to pre-alignment 5'-end quality trimming and sequence adapter clipping using Trimmomatic.
Cleaned-up reads were mapped to the hg19 reference genome using STAR (version 2.3.0), using splice aware alignments and allowing for 10 mismatches.
Intron-spanning read summarization was performed using a samtools-based filtering step and HTSeq (version 0.6.1) using union-mode, unstranded reads and minimal mapping quality of 35, all guided by the Ensembl gene annotation version 75.
Genome_build: hg19 (GRCh37)
Supplementary_files_format_and_content: allSamples_readCounts_ISreads.txt: Samples in the tab-delimited text file were processed according to the described protocol and data of all samples was merged in a comprehensive read count matrix.
|
| |
|
| Submission date |
May 21, 2018 |
| Last update date |
Nov 02, 2018 |
| Contact name |
Myron Best |
| E-mail(s) |
m.best@vumc.nl
|
| Organization name |
VU University Medical Center
|
| Department |
Neurosurgery
|
| Lab |
Neuro-oncology Research Group
|
| Street address |
De Boelelaan 1117
|
| City |
Amsterdam |
| ZIP/Postal code |
1081 HV |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE114710 |
Impact of Escherichia coli K12 and O18 on human platelets: effects on platelet activation, spliced platelet RNAs and proteins |
|
| Relations |
| BioSample |
SAMN09231852 |
| SRA |
SRX4106307 |
