|
| Status |
Public on Nov 03, 2024 |
| Title |
3DCS_4214 |
| Sample type |
SRA |
| |
|
| Source name |
blood
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: blood
cell type: PBMC
treatment: 3D hydrogel culture system
time: day 8
|
| Growth protocol |
Cells were suspended in 10% sucrose solution at 2x106 cells/mL and plated in PuraMatrix Peptide Hydrogel (Corning) diluted 1:1 with a 20% sucrose solution and supplemented with 100 ng/mL stem cell factor (SCF), 100 ng/mL Flt3 ligand (FLT3L), 20 ng/mL thrombopoietin (TPO), 2 ng/mL vascular endothelial growth factor (VEGF), 20 ng/mL IL-3, 20 ng/mL IL-6, 1 µmol/L StemRegenin1 (SR1), and 25 µg/mL Vitamin C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
3D hydrogel culture wells were washed with PBS with recovery of the hydrogel and cells by gentle pipetting in PBS buffer, then centrifuged for 5 minutes at 750 x g, then the hydrogel/PBMC pellet was resuspended in TrypLE solution and incubated at 37°C for 10 minutes. RPMI media with 10% FBS was added and centrifuged for 5 minutes at 750 x g. The resultant pellet was washed twice with PBS to remove hydrogel matrix debris. PBMCs were resuspended in PBS and passed through a 40 µM filter, pelleted by centrifugation at 300 x g for 5 minutes, and resuspended in RPMI media with 10% FBS. FACS was then used to isolate cells from any remaining hydrogel debris and recovered cells were concentrated to 1,000 cells/µl in RPMI media with 10% FBS for downstream scRNA-seq.
Single-cell RNA-seq library preparation was performed using the 10x Genomics Chromium platform with the Single Cell 3’ Library and Gel bead kit v3.1 following the manufacturer’s user guide.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
FASTQ files were generated using the Cell Ranger v6.0.1 (10X Genomics) bcl2fastq (Illumina) wrapper.
Sequencing reads were assembled and aligned against the GENCODE basic mouse reference using Cell Ranger v6.0.1 (10X Genomics) to generate count matrices.
Assembly: GRCh38
Supplementary files format and content: HDF5 gene count matrices
|
| |
|
| Submission date |
Oct 29, 2024 |
| Last update date |
Nov 04, 2024 |
| Contact name |
Jason Anthony Vander Heiden |
| E-mail(s) |
vandej27@gene.com
|
| Organization name |
Genentech
|
| Department |
Biological Research AI Development
|
| Street address |
1 DNA Way
|
| City |
South San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94080 |
| Country |
USA |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE280632 |
3D hydrogel culture system for generation of fibrosis-associated macrophages |
|
| Relations |
| BioSample |
SAMN44499888 |
| SRA |
SRX26538468 |
