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| Status |
Public on Jan 11, 2024 |
| Title |
Replicate 1 HTO |
| Sample type |
SRA |
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| Source name |
Neutrophils
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| Organism |
Mus musculus |
| Characteristics |
hashtag: TotalSeq™-A0301
cell type: Neutrophils
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| Treatment protocol |
Mice were administered intrapancreatic injections of FC1242L tumor cells. The mice were anaesthetized, the pancreas was exposed by surgical excision, and 10,000 FC1242L cels were re-suspended in 1X phosphate buffered saline (PBS) and mixed with Matrigel (BD) in a 1:1 ratio, and were injected as a volume of 50 µL into the body of the pancreas to form a visible bolus.
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| Growth protocol |
The luciferase-stable pancreatic ductal adenocarcinoma FC1242L cell line was generated as described in Leong et al., and was derived from the FC1242 cell line (kind gift from Dr Dannielle Engle, Tuveson lab, Cold Spring Harbour Laboratory) derived from Pdx1Cre; KrasG12D/+; Trp53R172H/+ (KPC) mice. FC1242L was culturecultured in complete media (cDMEM) comprising Dulbecco’s modified Eagle’s Medium (DMEM) (Gibco) supplemented with 10% fetal calf serum (FCS), 1% Penicillin-Streptomycin (Gibco) and seeded at a density of 2 X 10^5 cells/mL in T75 cell culture flask. Cells were grown at 37°C in 5% CO2 incubator until they reached 80% confluency. Upon reaching confluency, cells were trypsinized with pre-warmed 0.25% Trypsin with Ethylenediaminetetraacetic acid (EDTA), phenol red (Gibco) for 5 minutes at 37°C in 5% CO2 incubator, washed with pre-warmed cDMEM and counted, before seeding at a density of 2 X 10^5 cells/mL for the next passage.
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| Extracted molecule |
protein |
| Extraction protocol |
Tumor: At 6 weeks post injection, tumors were excised from tumor-bearing mice and weighed before mincing into smaller pieces using surgical scissors. Digestion mix (RPMI, 10% FCS, 500U/mL DNase I, 385U/mL collagenase D) was added to the minced tumor and incubated at 37°C for 30 minutes. Digestion enzymes were quenched with FACS buffer, and minced tumors were transferred into the gentleMACS™ C tubes (Miltenyi Biotec) for mechanical homogenization by the Xiril Dispomix using 2 rounds of Program 5 (20 seconds). Homogenized cells were filtered through 70-µm nylon mesh filter and washed with 20mL FACS buffer. Samples were then spun down at 400g for 5 minutes before RBC lysis with 1 X RBC lysis buffer for 3 minutes. Cells were washed filtered through a 70-µm nylon mesh filter. Blood: Murine blood was collected via cardiac puncture or incision in the submandibular region using a 5mm single-use lancet. 200 µL was transferred into an Eppendorf tube containing 20 µL of 0.5M EDTA. Blood samples were then lysed in 1 X red blood cell (RBC) lysis buffer (eBioscience) for 4 min. Samples were quenched with fluorescence-activated cell sorting (FACS) Buffer (1 X PBS, 4mM EDTA, 3% FCS) and spun down at 400g for 5 minutes. RBC lysis was repeated for a second time for another 2 minutes. Spleen: Spleens were excised from mice and single-cell suspensions were obtained by homogenizing spleen samples through a 70-µm nylon mesh filter with FACS buffer. Samples were then spun down at 400g for 5 minutes and lysed with 1 X RBC lysis buffer for 3 minutes. Cells were quenched with FACS buffer and filtered through a 70-µm nylon mesh filter. Bone marrow: A 23-gauge needle through the marrow cavity of the femur and the bone marrow was flushed out with 2 ml of FACS buffer using a 1ml syringe. For sorting purposes that required large cell numbers, both femur and tibia bones were used. Cells were then filtered and washed with FACS buffer, cells were spun down at 400g for 5 minutes. Sorting and hashtag labelling: Total neutrophils were sorted from the bone marrow, spleen, blood, and tumors from pancreatic tumor mice and identified as Lin-CD45+CD115-Ly6ClowSiglec-F-Gr1+CD11b+Ly6G+. Neutrophils were spun down and incubated with Totalseq-A anti-mouse Hashtag antibodies 1-8 at a concentration of 0.5ug per 100,000 cells for 30 minutes at 4°C. Cells were washed with FACS buffer, spun down, and resuspended in PBS with 1% bovine serum albumin.
Hashtagged neutrophils were counted and pooled, before used for input for the 10x Genomics 3' (v3) workflow following the manufacturer's instructions
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| Library strategy |
RNA-Seq |
| Library source |
other |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
10X Genomics
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| Data processing |
The quality of sequencing reads was evaluated using FastQC and MultiQC. Cell Ranger (version 2.2.0) was used to align the sequencing reads (fastq) to the mm10 mouse transcriptome and quantify the expression of transcripts in each cell. This pipeline resulted in a gene expression matrix for each sample, which records the number of UMIs for each gene associated with each cell barcode.
The gene expression matrix was subsequently analyzed in R using the Seurat package (4.0.5). Hashtags were demultiplexed using CITE-seq-Count. Doublets and multiplets were filtered out, as well as UMIs which had 2 or more hashtags associated with it, which resulted in 11, 682 remaining single cell transcriptomes. Additional QC included removing UMIs with excess mitochondrial reads (>5%), number of features less than 200 (low read counts) and more than 4200 (outliers).
After QC, the standard Seurat pipeline running NormalizeData(), FindVariableFeatures(selection.method =”vst”, nfeatures =2000), and ScaleData(). ScoreJackStraw() and JackstrawPlot was used to determine the number of dimensions before running RunPCA(), FindNeighbours(dims = 1:35), and FindClusters(resolution =0.8). RunUMAP(dim=1:35, n.neighbours =30) was used to visualize the data.
Assembly: mm10
Supplementary files format and content: Tab-separated barcodes and features file, and matrix files
Supplementary files format and content: Comma-separated metadata
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| Submission date |
Sep 18, 2023 |
| Last update date |
Jan 11, 2024 |
| Contact name |
Laiguan Ng |
| E-mail(s) |
Ng_Lai_Guan@immunol.a-star.edu.sg
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| Organization name |
Singapore Immunology Network
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| Street address |
8A Biomedical Grove IMMUNOS bldg, Level 3, SINGAPORE 138648
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| City |
Singapore |
| ZIP/Postal code |
138648 |
| Country |
Singapore |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE243466 |
Single cell RNA profiling of neutrophils in a model of pancreatic orthotopic cancer |
| GSE244536 |
Deterministic reprogramming of neutrophils in tumors |
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| Relations |
| BioSample |
SAMN37442698 |
| SRA |
SRX21810453 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7787587_Hashtag_table.txt.gz |
148 b |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
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