|
| Status |
Public on Jan 28, 2023 |
| Title |
Immune_p53 Off 2 |
| Sample type |
SRA |
| |
|
| Source name |
liver tumor
|
| Organism |
Mus musculus |
| Characteristics |
cell type: sorted primary immune cells (CD45+)
tissue: liver tumor
condition: orthotopic liver injection
treatment: p53 Off
sorting strategy: GFP-; Cd45+;DAPI-
mouse strain: C57Bl/6N
Sex: Female
|
| Treatment protocol |
Liver tumors were harvested from the indicated mouse models and processed for FACS using Collagenase IV (1 mg/mL) & Dispase II (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 immune cells from liver tumor (marked as GFP-negative, CD45-positive and DAPI-negative) were sorted using Sony 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 |
GFP-expressing NSP tumor cells were cultured off Dox to suppress p53 expression and orthotopically injected into liver of C57B/6 mice fed with Dox food. After 2-3 weeks tumor growth, the mice were randomized either keeping on Dox or removing Dox diet to control p53 expression in tumor cells
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Single-cell encapsulation and scRNA-seq library prep of Flow cytometry-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 40,000 cells at a final dilution of ~600 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.
scRNA-seq (3' kit, v 2.0)
|
| |
|
| 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 |
All scRNAseq data were processed into count matrices using 10x Genomics CellRanger All scRNAseq data were processed into count matrices using 10x Genomics CellRanger 6.0.0 with default parameters using reference mouse genome GRCm38/mm10 augmented with BioLegend TotalSeqB hashtag oligonucleotide barcode sequences for demultiplexing cellular compartments in downstream analysis oligonucleotide barcode sequences for demultiplexing cellular compartments in downstream analysis. Count matrices were processed to remove empty and low quality droplets by removing (in order): transcripts with more than 10 million or fewer than 100 total reads, droplets with library size greater than 150,000 or lower than 300 total read count, and droplets with fewer than 15 distinct expressed transcripts.
Potential doublets were removed using Solo version 1.2 (76) by training one doublet classification model per sample using default parameters for Solo and removing droplets using a threshold of 0.5. Subsequently, dead / dying cells were filtered by removing droplets with high mitochondrial RNA content (greater than 20% of transcript counts mapped to MT genes) or high ribosomal transcript count (greater than 15% of total transcript counts mapped to ribosomal genes). After pre-processing, poor quality samples with either low numbers of recovered cells or low number of distinct transcripts recovered were removed.
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files
|
| |
|
| Submission date |
Jan 27, 2023 |
| Last update date |
Jan 28, 2023 |
| Contact name |
Yu-Jui Ho |
| E-mail(s) |
hoy@mskcc.org
|
| Organization name |
Memorial Sloan Kettering Cancer Center
|
| Department |
Cancer Biology & Genetics Program
|
| Street address |
417 E 68th St
|
| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10065 |
| Country |
USA |
| |
|
| Platform ID |
GPL24247 |
| Series (2) |
| GSE223874 |
Senescence rewires microenvironment sensing to facilitate anti-tumor immunity (scRNA-Seq) |
| GSE223875 |
Senescence rewires microenvironment sensing to facilitate anti-tumor immunity |
|
| Relations |
| BioSample |
SAMN32941034 |
| SRA |
SRX19199665 |
