 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on May 10, 2021 |
| Title |
TNF_P2 |
| Sample type |
SRA |
| |
|
| Source name |
murine macrophage-like cell line
|
| Organism |
Mus musculus |
| Characteristics |
cell type: macrophage
treatment: treated with WBP and TNF-α for 24 hours
strain: BALB/c
|
| Growth protocol |
The murine macrophage-like cell line (RAW264.7) was obtained from Cell Resource Center, IBMS, CAMS/PUMC (Beijing, China) and cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum and antibiotics (100 U/mL penicillin and 100 U/mL streptomycin) at 37 °C in a humidified atmosphere containing 95% O2 and 5% CO2 atmosphere.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using RNeasy mini kit (Qiagen, Germany) according to the manufacturer’s protocol. RNA integrity was evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The quantity of total RNA samples was measured using Qubit®3.0 Fluorometer (Thermo Fisher Scientific, USA) and NanoDrop One (Wilmington, DE, USA).
Paired-end libraries were synthesized by using the TruSeq™ RNA Sample Preparation Kit (Illumina, USA) following TruSeq™ RNA Sample Preparation Guide. Briefly, the poly-A containing mRNA molecules were purified using poly-T oligo-attached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under 94℃ for 8 min. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. This is followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then go through an end repair process, the addition of a single ‘A’ base, and then ligation of the adapters. The products are then purified and enriched with PCR to create the final cDNA library. Purified libraries were quantified by Qubit® 2.0 Fluorometer (Life Technologies, USA) and validated by Agilent 2100 bioanalyzer (Agilent Technologies, USA) to confirm the insert size and calculate the mole concentration. Cluster was generated by cBot with the library diluted to 10 pM and then were sequenced on the Illumina NovaSeq 6000 (Illumina, USA). The sequencing work was undertaken by Beijing Zhimei Yinuo Biotechnology Co., Ltd (Beijing, China).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Briefly, The poly-A containing mRNA molecules were purified using poly-T oligo-attached magnetic beads.
Following purification, the mRNA is fragmented into small pieces using divalent cations under 94℃ for 8 min. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers.
This is followed by second strand cDNA synthesis using DNA Polymerase I and RNase H.
These cDNA fragments then go through an end repair process, the addition of a single ‘A’ base, and then ligation of the adapters.
The products are then purified and enriched with PCR to create the final cDNA library.
Purified libraries were quantified by Qubit® 2.0 Fluorometer (Life Technologies, USA) and validated by Agilent 2100 bioanalyzer (Agilent Technologies, USA) to confirm the insert size and calculate the mole concentration. Cluster was generated by cBot with the library diluted to 10 pM and then were sequenced on the Illumina NovaSeq 6000 (Illumina, USA).
Supplementary_files_format_and_content: Expression value of genes and transcripts
|
| |
|
| Submission date |
Jan 21, 2021 |
| Last update date |
May 10, 2021 |
| Contact name |
Dan Wu |
| E-mail(s) |
wudan971111@outlook.com
|
| Organization name |
Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences
|
| Street address |
No. 16, Nei Nanxiao Street, Dongzhimen, Dongcheng District
|
| City |
Beijing |
| ZIP/Postal code |
100010 |
| Country |
China |
| |
|
| Platform ID |
GPL24247 |
| Series (1) |
| GSE165272 |
Integrative Pharmacology Strategy Powers the Delineation of the Main Active Components and Exploration the Underlying Mechanism of WBT Acting on Rheumatoid Arthritis |
|
| Relations |
| BioSample |
SAMN17485421 |
| SRA |
SRX9911374 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
