|
| Status |
Public on Dec 26, 2019 |
| Title |
Single cells UP-8165PV 24wk |
| Sample type |
SRA |
| |
|
| Source name |
Glioblastoma organoid
|
| Organism |
Homo sapiens |
| Characteristics |
culture duration: 24 weeks
|
| Treatment protocol |
Multiple tumor pieces and GBOs were dissociated using a papain-based neural tissue dissociation kit (Miltenyi Biotech). Crude dissociates from parental tumor samples were treated with ammonium chloride based RBC lysis buffer for 5 minutes. All samples were washed three times by centrifugation at 200 g for 5 minutes and resuspension in 10 mL of calcium-free, magnesium-free DPBS. Cells were strained through a 70 µm filter (Miltenyi Biotech), analyzed for viability by trypan blue staining, and counted using an automatic cell counter.
|
| Growth protocol |
Glioblastoma organoids were generated by mincing fresh tumor tissue and cultured on an orbital shaker
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Drop-seq was performed as previously described with minor modifications (Macosko et al., 2015). Microfluidic chips were obtained from FlowJEM with plasma bonding and aquapel treatment using the same design as previously published. Each droplet co-encapsulation run was performed with 50% additional reagent volume to account for syringe and tubing dead volume as well as occasional microfluidic channel clogging and re-starts. Each sample was loaded onto 2-4 droplet generation runs, and cell suspensions and droplets were kept on ice prior to droplet encapsulation and droplet breakage. In the cDNA PCR amplification stage, between 4,000-8,000 beads were combined in a single PCR tube reaction and purified individually using 0.6x SPRI beads. cDNA across multiple runs of the same sample were pooled together and used as input for tagmentation, where the tagment reaction time was increased to 8 minutes. Final samples were purified once with 0.6x SPRI beads and a second time with 1.0x SPRI beads. Sequencing library fragment sizes were quantified by bioanalyzer, and concentrations were quantified by qPCR.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
| |
|
| Data processing |
Raw sequencing data was demultiplexed with bcl2fastq2 with adapter trimming turned off. Additional processing was performed using drop-seq tools v.2.1.0 with GRCh38 as the reference genome, and gencode v.28 GTF was used as the annotation file.
Genome_build: GRCh38
Supplementary_files_format_and_content: Gene counts matrix was constructed using drop-seq tools v2.1.0
|
| |
|
| Submission date |
Dec 12, 2019 |
| Last update date |
Dec 26, 2019 |
| Contact name |
Daniel Zhang |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Pennsylvania
|
| Street address |
3400 Civic Center Boulevard
|
| City |
Philadelphia |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL21697 |
| Series (2) |
| GSE141946 |
Single Cell RNA-seq of Glioblastoma Tumors and GBOs |
| GSE141947 |
Glioblastoma Tumors and GBOs |
|
| Relations |
| BioSample |
SAMN13551699 |
| SRA |
SRX7354971 |
