|
| Status |
Public on Aug 05, 2022 |
| Title |
PBMCs_3 with ConA and ALA |
| Sample type |
SRA |
| |
|
| Source name |
peripheral blood mononuclear cells
|
| Organism |
Canis lupus familiaris |
| Characteristics |
cell type: peripheral blood mononuclear cells
treatment: treated with ConA and ALA
|
| Treatment protocol |
PBMCs were treated with ConA (0.5 μg/ml) alone or ALA (1.0 mM) alone or both reagents for 24 hours before being harvested.
|
| Growth protocol |
Peripheral blood mononuclear cells (PBMCs) were isolated from healthy specific pathogen-free male beagles (maintained as blood donors in our veterinary teaching hospital). PBMCs were isolated by density gradient centrifugation with Lymphoprep (Axis-Shield, Olso, Norway) and cultured in the RPMI1640-based complete medium (RPMI1640 (FUJIFILM Wako Pure Chemical Corporation), 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 55 μM 2-mercaptoethanol).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from the PBMCs using the Maxwell RSC simplyRNA cells kit (Promega). Polyadenylated mRNA was purified with oligo dT beads (NEBNext Poly (A) mRNA magnet Isolation Module, New England Biolabs, NEB). Complementary DNA (cDNA) libraries for Illumina sequencing were generated with NEBNext Ultra II RNA library Prep kit (NEB) and NEBNext Multiplex Oligos for Illumina (NEB).
Extracted mRNA was incubated with NEBNext Random Primers in NEBNext First Strand Synthesis Reaction Buffer at 94°C for 15 min, and reverse transcription was performed with NEBNext Strand Synthesis Enzyme Mix. After reverse transcription, index sequences were inserted to cDNA fragments with PCR amplification using primers included in NEBNext Multiplex Oligos for Illumina. Following purification with AMPureXP beads (Beckman Coulter), the quality and the concentration of the libraries was evaluated using a Bioanalyzer. The confirmed libraries were mixed to equal molecular amounts against the clustering and sequencing on an Illumina NextSeq 500 DNA sequencer with 75 bp paired-end sequencing kit (Illumina).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
BCL files produced by Illimina NextSeq 500 were converted to FASTQ files using bcl2fastq tool. Created FASTQ files were imported to CLC Genomics Workbench software (ver.20.0.4).
Reads with more than 2 ambiguous nucleotides and reads with quality scores less than 20 as calculated by the Phred program were removed using CLC Genomics Workbench software (ver.20.0.4). Long reads with more than 1000 nucleotides and short reads with fewer than 20 nucleotides were also discarded.
The trimmed reads were mapped to the human reference genome CanFam3.1 release-98 using CLC Genomics Workbench software (ver.20.0.4) with default setting.
Reads Per Kilobase of exon per Million mapped reads (RPKM) and Transcripts Per Million (TPM) were calculated using CLC Genomics Workbench software (ver.20.0.4).
Assembly: CanFam3.1
Supplementary files format and content: tab-delimited text files include RPKM and TPM values for each sample.
|
| |
|
| Submission date |
Jun 08, 2022 |
| Last update date |
Aug 05, 2022 |
| Contact name |
Yoichi Mizukami |
| Organization name |
Yamaguchi university
|
| Department |
Science Research Center
|
| Lab |
Institute of Gene Research
|
| Street address |
Minamikogushi1-1-1
|
| City |
Ube |
| State/province |
Yamaguchi |
| ZIP/Postal code |
7558505 |
| Country |
Japan |
| |
|
| Platform ID |
GPL21400 |
| Series (1) |
| GSE205679 |
The effect of 5-aminolevulinic acid on canine peripheral blood mononuclear cells |
|
| Relations |
| BioSample |
SAMN28921054 |
| SRA |
SRX15631359 |
