|
| Status |
Public on Apr 27, 2023 |
| Title |
Epithelial_K6_4.1 |
| Sample type |
SRA |
| |
|
| Source name |
pancreas
|
| Organism |
Mus musculus |
| Characteristics |
cell type: sorted primary pancreatic epithelial cells (mKate2+)
tissue: pancreas
condition: K6
model: KPC;RIK
alleles: p48Cre;RIK;LSL-KrasG12D;p53fl/wt
treatment: PDAC lesion (Liver Metastases)
sorting strategy: mKate2+;Cd45-;DAPI-
time point_(analyses): PDAC lesion
Sex: Male
|
| Treatment protocol |
Pancretata were harvested from the indicated mouse models and processed for FACS sorting using Collagenase (1 mg/mL) & Dispase (2 U/mL) mix digestion solution and Gentle-MACS dissociator (mTDK_1 protocol), followed by a 0.05% Trypsin-EDTA step. Cell suspensions were incubated with Fc block and CD45-APC or CD45-APC-Cy7 antibody to mark immune cells. Live pancreatic epithelial cells (marked as mKate2-positive, CD45-negative and DAPI-negative) or immune cells (marked as mKate2-negative, CD45-positive and DAPI-negative) were sorted using Aria3 or Aria 1 sorter, and collected in 2% 1x PBS buffer containing 2% fetal bovine serum (FBS). Single-cell suspensions were spun down and resuspended in 0.05% BSA containing 1 U/µL Invitrogen Ambion RNase Inhibitor (AM2682, Thermo Fisher Scientific).
|
| Growth protocol |
When indicated, 5 weeks-old KC;RIK (p48-Cre;RIK;LSLKrasG12D) littermate male mice were treated with 8 hourly intraperitoneal injections of 80 μg/kg caerulein (Bachem) for 2 consecutive days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Single-cell encapsulation and scRNA-seq library prep of FACS-sorted cell suspensions was performed on the Chromium instrument (10x Genomics) following the user manual (Reagent Kit 3’ v2). Each sample loaded onto the cartridge contained approximately 5,000 cells at a final dilution of ~500 cells/μl. Transcriptomes of encapsulated cells were barcoded during reverse transcription.
cDNA was purified with DynaBeads, followed by amplification per the user manual (Reagent Kit 3’ v2). Next, the PCR-amplified product was fragmented, A-tailed, purified with 1.2X SPRI beads, ligated to sequencing adapters and indexed by PCR. Indexed DNA libraries were double-size purified (0.6–0.8X) with SPRI beads.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
10x Genomics (3' kit, v2.0)
|
| Data processing |
The demultiplexing, barcoded processing, alignment, and count matrix generation was done using the SEQC software.
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files.
Supplementary files format and content: ProgressionCohort.h5ad: AnnData file containing processed scRNA-seq counts for the entire epithelial Progression Cohort. Counts are normalized but un-logged, and raw data is available in the .raw attribute.
Supplementary files format and content: Combined_ImmuneEpi.h5ad: AnnData file containing processed scRNA-seq counts for the entire epithelial and immune Progression Cohort. Counts are normalized but un-logged.
Supplementary files format and content: PerturbationCohort_K3.h5ad: AnnData file containing processed scRNA-seq counts for epithelial cells from K3 condition with and without IL-33 perturbation. Counts are log-normalized.
|
| |
|
| Submission date |
Jul 11, 2022 |
| Last update date |
May 10, 2023 |
| Contact name |
Cassandra Burdziak |
| E-mail(s) |
burdziac@mskcc.org
|
| Organization name |
Memorial Sloan Kettering Cancer Center
|
| Street address |
417 E 68th St
|
| City |
New York City |
| State/province |
New York |
| ZIP/Postal code |
10065 |
| Country |
USA |
| |
|
| Platform ID |
GPL24247 |
| Series (2) |
| GSE207938 |
Epigenetic plasticity cooperates with emergent cell-cell interactions to drive neoplastic tissue remodeling in the pancreas [scRNA-seq] |
| GSE207943 |
Epigenetic plasticity cooperates with emergent cell-cell interactions to drive neoplastic tissue remodeling in the pancreas |
|
| Relations |
| BioSample |
SAMN29635559 |
| SRA |
SRX16119906 |
