|
| Status |
Public on Jul 01, 2017 |
| Title |
SW1990 positive rep3 |
| Sample type |
SRA |
| |
|
| Source name |
pancreatic adenocarcinoma cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: pancreatic adenocarcinoma
cell line: SW1990
gctm5: positive
|
| Treatment protocol |
All cells were trypsinized and resuspended in GCTM-5 IgG1 antibody supernatant (primary antibody). The cells were incubated for 1 hour at 4°C, then washed thrice with FACS buffer (0.1% BSA, 2 mM EDTA in PBS) and subsequently incubated with the secondary antibody (goat anti-mouse HRP) for 30 minutes. Cells were washed thrice and resuspended at 2.E6 cells/ml for sorting. Flow cytometry was performed using BD FACSAria™ Fusion flow cytometer (BD Biosciences) and data was analysed by BD FACSDiva software provided with the system. The gates were chosen to include only highly positive and negative cell populations and the sorted cells were then used for RNA extraction.
|
| Growth protocol |
CFPAC-1 and SW-1990 cells were grown as monolayer cultures in Iscove’s Modified Dulbecco’s Medium supplemented with 10% fetal bovine serum and 2 mM L-glutamine. Cells were passaged every 3-4 days upon reaching 70-80% confluence, using TrypLE (Gibco) for dissociation, and were subcultured in a split ratio of 1:5.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the RNeasy® Mini Kit (Qiagen, 74104), and purified with DNase I (Qiagen, 79254). RNA was then quantified using the NanoDrop2000 spectrophotometer (Thermo Scientific). 1μg quantified total RNA was taken and enriched for PolyA using Oligo(dT) Microparticles.
Transcriptome library for sequencing was constructed according to SureSelect Strand-Specific RNA Library Prep Kit protocol outlined in “SureSelect Strand-Specific RNA Library Prep for Illumina Multiplexed Sequencing” (Cat #5500-0135). Briefly, the mRNA was fragmented for 2 minutes at elevated temperature (94oC) in the presence of divalent cations and first strand cDNA was synthesized. The single stranded cDNA was cleaned up using HighPrep (Magbio, Cat #AC-60050). Strand Specificity is maintained by the addition of Actinomycin D at this step. Second strand cDNA was synthesized and end repaired using Second Strand Synthesis + End Repair mix. The cDNA was cleaned up using HighPrep (Magbio, Cat #AC-60050). Adapters were ligated to the cDNA molecules after addition of “A”- base. HighPrep cleanup was performed post ligation. The library was indexed and enriched for adapter ligated fragments using 10 cycles of PCR. The prepared library was quantified using Qubit and validated for quality by running an aliquot on D1000 Tapes. Finally, the adapter positive fragments were quantified using KAPA qPCR quantification kit (KAPA Biosystems; Cat #KK4824).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
Base-calling was done using bcl2fastq2 Conversion Software v2.17.
Sequenced reads were trimmed for adaptor sequence, and mapped to hg19/GRCh37 whole genome using QuasR with parameters -m 1 --best --strata -v 2
Reads Per Kilobase of gene per Megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009.
Genome_build: hg19/GRCh37
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample
|
| |
|
| Submission date |
Feb 22, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Kouichi Hasegawa |
| E-mail(s) |
hasegawa-k@sahs.med.osaka-u.ac.jp
|
| Organization name |
Osaka University
|
| Department |
StemRIM Institute of Regeneration-Inducing Medicine
|
| Lab |
Drug Efficacy Evaluation & Pharmacology Dept. StemRIM
|
| Street address |
6F Techno Alliance Bldg. 2-8 Yamadaoka
|
| City |
Suita |
| State/province |
Osaka |
| ZIP/Postal code |
565-0871 |
| Country |
Japan |
| |
|
| Platform ID |
GPL20301 |
| Series (1) |
| GSE95176 |
GCTM-5 positive and negative cells in pancreatic adenocarcinoma cell lines |
|
| Relations |
| BioSample |
SAMN06368686 |
| SRA |
SRX2581687 |
