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Status |
Public on Mar 06, 2022 |
Title |
S04 |
Sample type |
SRA |
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Source name |
Unstimulated_8h_dog cell line 030-D
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Organism |
Canis lupus |
Characteristics |
cell line: dog cell line 030-D stimulus: Unstimulated time: 8h replicate: 1
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Treatment protocol |
4 x 10^6 030-D cells were stimulated with inactivated whole-cell preparations of L. interrogans serogroup Canicola or Icterohaemorrhagiae, excipient (non-antigen control) and two bivalent Leptospira vaccines.
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Growth protocol |
030-D cell line, derived from a canine malignant histiocytosis, was cultured at 37°C and 5% CO2 in RPMI-1640 (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco), 100 U/ml penicillin, 100 µg/ml streptomycin and 300 ng/ml L-glutamine (all Gibco).
|
Extracted molecule |
polyA RNA |
Extraction protocol |
Cells were harvested and centrifuged 5 min at 300g. Pellet was suspended in 200 µl PBS and lysed in 550 µl Lysis/binding buffer (Roche) supplemented with 1% DTT (Sigma Aldrich). Cell lysates were stored at -80°C until RNA isolation with the MagNA Pure LC RNA isolation kit-High performance (Roche) on the MagNA Pure LC 2.0 instrument following the manufacturer’s protocol. Isolated RNA samples were quantified using UV spectroscopy (Take3 module, Synergy Mx, BioTek, Winooski, USA). RNA integrity was analysed by agarose gel electrophoresis using 2% E-Gel EX pre-stained gels (Thermo Fischer Scientific). Library preparation and RNA sequencing was performed at MAD: Dutch Genomics Service & Support (University of Amsterdam, The Netherlands). Libraries were sequenced using an NextSeq 550 platform and 20 million reads per sample.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
NextSeq 550 |
|
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Data processing |
Only the first read of the paired-end reads were used for data analysis, these were treated as single reads by our fastq file processing pipeline. The quality of reads was assessed using FastQC version 0.11.5 Trimmed read sequences were mapped to the dog genome using STAR (version 2.5.3a) The number of mapped reads were counted for each gene and compiled into an expression matrix using featureCounts (version 1.6.0) Genome_build: CanFam3.1, release 97 Supplementary_files_format_and_content: Countdata.xlsx: count data per sample
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Submission date |
Jan 03, 2022 |
Last update date |
Mar 06, 2022 |
Contact name |
Jeroen Pennings |
E-mail(s) |
Jeroen.Pennings@rivm.nl
|
Phone |
+31 88 689 2214
|
Organization name |
Natl. Inst. Public Health & Environment
|
Street address |
A. van Leeuwenhoeklaan 9
|
City |
Bilthoven |
ZIP/Postal code |
3721MA |
Country |
Netherlands |
|
|
Platform ID |
GPL31163 |
Series (1) |
GSE192945 |
Biomarkers for canine Leptospira vaccine potency |
|
Relations |
BioSample |
SAMN24588246 |
SRA |
SRX13584807 |