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| Status |
Public on Nov 01, 2023 |
| Title |
PDX, vehicle, rep 2 |
| Sample type |
SRA |
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| Source name |
PDX
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| Organism |
Homo sapiens |
| Characteristics |
tissue: PDX
cell type: PDX
treatment: vehicle
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| 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).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
PDX_CountMatrix_dds.Rdata
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| Data processing |
PDXes and autochthonous KP LUAD tumors were FACS-isolated, RNA was extracted, and libraries were prepared for SMARTerSeq2, n = 3 mice/group. FASTQ files were processed using the standard DESeq2 pipeline (version 1.24.0). Reads were aligned to the GRCm38 mouse genome. Count matrices were generated was performed using DESeq2. Data were filtered to only include genes with 10 or more counts in 2 or more samples. The significance of differential expression between vehicle and treatment groups was quantified by one-way ANOVA.
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| Submission date |
Oct 28, 2023 |
| Last update date |
Nov 01, 2023 |
| Contact name |
ZHUXUAN LI |
| E-mail(s) |
liz3@mskcc.org
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| Phone |
6467043994
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| Organization name |
Memorial Sloan Kettering Cancer Center
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| Department |
Cancer Biology and Genetics
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| Lab |
Tuomas Tammela
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| Street address |
417 E 68th St
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10065 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE246480 |
Alveolar differentiation drives resistance to KRAS inhibition in lung adenocarcinoma [RNA-seq] |
| GSE246482 |
Alveolar differentiation drives resistance to KRAS inhibition in lung adenocarcinoma |
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| Relations |
| BioSample |
SAMN38027486 |
| SRA |
SRX22256174 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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