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| Status |
Public on Dec 13, 2018 |
| Title |
pbmc_gex |
| Sample type |
SRA |
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| Source name |
PBMCs from a healthy donor
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| Organism |
Homo sapiens |
| Characteristics |
cell type: PBMCs from a healthy donor
condition: Cryopreserved
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| Growth protocol |
Tumor tissues were minced finely, washed twice in ADMEM/F12 (Invitrogen) containing 1X Normocin (InvivoGen), resuspended in Type I collagen gel (Trevigen), and layered in a double dish air-liquid culture system as previously described8. Organoids were cultured in human organoid medium (ADMEM/F12 supplemented with 50% Wnt3a, RSPO1, Noggin-conditioned media (L-WRN, ATCC), HEPES (1mM, Invitrogen), Glutamax (1X, Invitrogen), Nicotinamide (10 mM, Sigma), N-Acetylcysteine (1 mM, Sigma), B-27 without vitamin A (1X, Invitrogen), A83-01 (0.5 μM, Tocris), Pen-Strep Glutamine (1X, Invitrogen), Gastrin (10 nM, Sigma), SB-202190 (10 μM, Sigma), and EGF (50 ng/mL, Invitrogen). Organoids were passaged every 14-30 days by dissociation with 200 units ml−1 collagenase IV (Worthington) at 37 °C for 30 min, followed by three 5-min washes with 100% FBS and replating at a 1:4 split into new air-liquid interface collagen gels. Additionally, in some cases, media was supplemented with recombinant human IL-2 (Peprotech) at 600 or 6000 IU/ml.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Tumor tissues were obtained through the Stanford Tissue Bank from patients undergoing surgical resection at Stanford University Medical Center (SUMC). All experiments utilizing human material were approved by SUMC’s Institutional Review Board and performed under protocol #28908. Written informed consent for research was obtained from donors prior to tissue acquisition. Samples were confirmed to be tumor by pathological assessment at SUMC.
Cellular suspensions were loaded on a ChromiumTM Single Cell Controller instrument (10x Genomics, Pleasanton, CA, USA) to generate single-cell GEMs. Single-cell RNA-Seq libraries were prepared using the ChromiumTM Single Cell 5’ Library & Gel Bead Kit (P/N 1000006, 10x Genomics). GEM-RT was performed in a C1000 Touch Thermal cycler with 96-Deep Well Reaction Module (Bio-Rad; P/N 1851197): 53 °C for 45 min, 85 °C for 5 min; held at 4 °C and stored at -20 °C. The GEMs were shipped to 10x Genomics on dry ice, then broken and the single-strand cDNA was cleaned up with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific; P/N 37002D) . Barcoded, full length cDNA was amplified using the C1000 Touch Thermal cycler with 96-Deep Well Reaction Module: 98 °C for 45 s; cycled 13 ×: 98 °C for 20 s, 67 °C for 30 s, and 72 °C for 1 min; 72 °C for 1 min; held at 4 °C. Amplified cDNA product was cleaned up with the SPRIselect Reagent Kit (0.6 × SPRI; Beckman Coulter; P/N B23318) . Barcoded, full-length V(D)J segments were enriched from amplified cDNA with primers specific to TCR constant regions. The target enrichment 1 was performed in a C1000 Touch Thermal cycler with 96-Deep Well Reaction Module: 98 °C for 45 s; cycled 10 ×: 98 °C for 20 s, 67 °C for 30 s, and 72 °C for 1 min; 72 °C for 1 min; held at 4 °C. The target enrichment 1 was cleaned up with the SPRIselect Reagent Kit (0.8 × SPRI). The target enrichment 2 was performed in a C1000 Touch Thermal cycler with 96-Deep Well Reaction Module: 98 °C for 45 s; cycled 10 ×: 98 °C for 20 s, 67 °C for 30 s, and 72 °C for 1 min; 72 °C for 1 min; held at 4 °C. The target enrichment 2 was cleaned up twice with the SPRIselect Reagent Kit (0.5 × and 0.8 × SPRI). The enrichment primers can be found in Supplementary Table 4. 5’ gene expression and enriched libraries were constructed using the reagents in the ChromiumTM Single Cell 3’/5’ Library Construction kit (P/N 1000020). For 5’ gene expression library construction, these steps were followed: (1) fragmentation, end repair and A-tailing; (2) post fragmentation, end repair and A-tailing cleanup with SPRIselect; (3) adaptor ligation; (4) post ligation cleanup with SPRIselect; (5) sample index PCR and cleanup. For the enriched library construction, these steps were followed: (1) fragmentation, end repair and A-tailing; (2) adaptor ligation; (3) post ligation cleanup with SPRIselect; (4) sample index PCR and cleanup. The barcode sequencing libraries were quantified by quantitative PCR (KAPA Biosystems Library Quantification Kit for Illumina platforms P/N KK4824). Sequencing libraries were loaded at concentrations on sequencers with the read configuration as specified in Table S5.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Single cell 5' RNA-seq
PBMC_Healthy_Donor_GEX
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| Data processing |
The Cell Ranger (CR) Software Suite (version 2.1.0) was used for 5’ gene counting and V(D)J sequence assembly and paired clonotype calling.
Genome_build: GRCh38
Supplementary_files_format_and_content: csv, tsv, mtx files
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| Submission date |
Mar 02, 2018 |
| Last update date |
Dec 13, 2018 |
| Contact name |
Calvin Kuo |
| E-mail(s) |
[email protected]
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| Phone |
6504985171
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| Organization name |
Stanford University
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| Department |
Department of Medicine, Division of Hematology
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| Street address |
265 Campus Dr.
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| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE111360 |
Organoid modeling of the tumor immune microenvironment |
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| Relations |
| BioSample |
SAMN08630702 |
| SRA |
SRX3757650 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3029119_pbmc_gex.barcodes.tsv.gz |
2.2 Mb |
(ftp)(http) |
TSV |
| GSM3029119_pbmc_gex.genes.tsv.gz |
258.6 Kb |
(ftp)(http) |
TSV |
| GSM3029119_pbmc_gex.matrix.mtx.gz |
24.2 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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