 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Nov 01, 2023 |
| Title |
subq,MRTX,BFPpos,HTO |
| Sample type |
SRA |
| |
|
| Source name |
subcutanous tumors
|
| Organism |
Mus musculus |
| Characteristics |
tissue: subcutanous tumors
cell line: KP tumor cells
cell type: primary lung tumor cells
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
To dissociate tumors into single-cell suspensions, primary tumors were finely chopped with scissors and incubated with digestion buffer containing collagenase IV (#17104019, ThermoFisher Scientific, 0.1 U/ml), dispase (#354235, Corning, 0.6 U/ml), and DNase I (#69182–3; Sigma Aldrich, 10 U/ml). For dissociating normal lung epithelial cells, digestion buffer was administrated intratracheally into the lung as described before (27). Following enzymatic dissociation, samples were washed with 2% heat-inactivated FBS in S-MEM (#11380037, Thermo Fisher Scientific), filtered through a 100 μm cell strainer (#431752, Corning), and centrifuged at 1500 rpm for 5 min at 4 °C. The supernatant was removed, and the pellet was resuspended in lysis buffer (#555899, BD Biosciences) to remove red blood cells. Cells were passed through a 40 μm strainer (#431750, Corning) and centrifuged at 1500 rpm for 5 min at room temperature. Cells were resuspended in 2% of heat-inactivated FBS in PBS. Cell suspensions were blocked for 5 min at 4 °C with rat anti-mouse CD16/CD32 (#553142, Mouse BD Fc Block, BD Biosciences) in FACS buffer, and incubated for 30 min with a mix of four APC-conjugated antibodies binding CD45 (#103112, Biolegend), CD31 (#102410, Biolegend), CD11b (#101212, Biolegend), F4/80 (#123116, BioLegend), CD19 (#115512, Biolegend), and TER-119 (#116212, Biolegend). 300 nM DAPI was added as a live-cell marker. Individual cancer cell suspensions were incubated for 30 min with hashtag oligonucleotide-conjugated antibodies in addition to FACS antibodies.
Sorted cell suspensions were prepared for scRNA-seq using the 3′ v3 10X Genomics Chromium platform according to manufacturer’s instructions. Briefly, sorted cells were washed once with PBS containing 1% bovine serum albumin (BSA) and resuspended in PBS containing 1% BSA to a final concentration of 700–1,300 cells per μl. The viability of cells in all experiments was above 80%, as confirmed with 0.2% (w/v) Trypan Blue staining (Countess II, Invitrogen). Cells were captured in droplets. Following reverse transcription and cell barcoding in droplets, emulsions were broken, and cDNA purified using Dynabeads MyOne SILANE followed by PCR amplification as per manufacturer’s instructions. Between 20,000 to 25,000 cells were targeted for each droplet formulation lane. Samples were multiplexed together in the lanes following the TotalSeq B cell hashing protocol. Final libraries were sequenced on Illumina NovaSeq S4 platform (R1 – 28 cycles, i7 – 8 cycles, R2 – 90 cycles).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
10x
|
| Data processing |
FASTQ files of single-cell RNA-sequencing data generated on the 10X Chromium platform were processed using the standard CellRanger pipeline (version 5.0.0). Reads were aligned to a custom GRCm38 / mm10 reference including the tdTomato, GFP, and mKate transgenes used in this study. Cell-gene count matrices were analyzed using a combination of published packages and custom scripts centered around the scanpy / AnnData ecosystem. Single-cell RNA-sequencing datasets from GFPshRNA KP LUAD tumors, MRTX1133-treated KP LUAD tumors, and lung regeneration experiments were analyzed separately using the same workflows. Single-cell RNA-sequencing data from treated and control replicates were dehashed and compiled into a combined count matrix. Cells with less than 500 UMIs, more than 20% mitochondrial UMIs, and low complexity based on the number of detected genes vs. number of UMIs were removed. Doublets were filtered using scrublet. UMI counts were normalized using the size factor approach described by Lun et al. . Highly variable features were selected using a variance stabilizing transformation and dimensionality reduction was performed on normalized, log2-transformed count data using principal component analysis. The dimensionality reduced count matrices were used as input for UMAP-embedding and unsupervised clustering with the leiden algorithm; bbknn was used to control for batch effects.
Supplementary files format and content: mm10
Supplementary files format and content: matrix files
|
| |
|
| Submission date |
Oct 28, 2023 |
| Last update date |
Nov 01, 2023 |
| Contact name |
ZHUXUAN LI |
| E-mail(s) |
liz3@mskcc.org
|
| Phone |
6467043994
|
| Organization name |
Memorial Sloan Kettering Cancer Center
|
| Department |
Cancer Biology and Genetics
|
| Lab |
Tuomas Tammela
|
| Street address |
417 E 68th St
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10065 |
| Country |
USA |
| |
|
| Platform ID |
GPL24247 |
| Series (2) |
| GSE246481 |
Alveolar differentiation drives resistance to KRAS inhibition in lung adenocarcinoma [scRNA-seq] |
| GSE246482 |
Alveolar differentiation drives resistance to KRAS inhibition in lung adenocarcinoma |
|
| Relations |
| BioSample |
SAMN38027498 |
| SRA |
SRX22256249 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7869604_adataFinal_ZL-2552_HOPX_LT1_10D_BFPplus_fixed_index.h5ad.gz |
82.0 Kb |
(ftp)(http) |
H5AD |
SRA Run Selector |
| Raw data are available in SRA |
|
|
|
|
 |
