2024-04-16
ENA first public
ERC000011
ENA-CHECKLIST
Single Cell Discoveries B.V
INSDC center name
public
INSDC status
ERS18638330
SRA accession
E-MTAB-13968:LXFS human
Submitter Id
6
week
age
ArrayExpress
broker name
lung cancer cell
cell_type
not collected
collection date
human
common name
Protocols: This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Society of Laboratory Animals (GV SOLAS) in an AAALAC accredited animal facility. All animal experiments were approved by the Committee on the Ethics of Animal Experiments of the regional council (Permit Numbers: I-19/02). Non-small cell lung cancer (NSCLC) PDX models were implanted subcutaneously in 4–6-week-old female NMRI nude mice (Charles River, Germany) under isoflurane anesthesia. Tumor growth was determined by a two-dimensional measurement with calipers twice a week. Different analyses of the PDX models were performed when tumor size reached 400 – 500 mm³. Animals were sacrificed and tumors were sampled for subsequent analysis.  Cells were processed according to the 10x protocol for the fixation of cells and nuclei. Briefly, suspensions of up to 10 million cells were spun-down at 350 rcf for 5 min at 4C and cells were resuspended in 1 mL of Fixation Buffer (4% Formaldehyde, 1X Conc. Fix and Perm Buffer – 10x Genomics PN-2000517). Cells were fixed for 16-24h at 4C. To stop the fixation, cells were spun-down at 850 rcf for 5 min at room temperature and quenched with 1 mL of Quenching Buffer (1X Conc. Quench Buffer – 10x Genomics PN-2000516). Preparations were then processed by adding 0.1 volumes of Enhancer (10x Genomics PN-2000482) and 10% glycerol for long-term storage and shipment to Single-cell Discoveries B.V..  Samples were then thawed at room temperature, centrifuged at 850 rcf for 5 min and resuspended in 1 mL 0.5X PBS - 0,02% BSA. Cell concentration and viability was then assessed by AO-PI staining with a LUNA-FX7™ Automated Cell Counter (Logos Biosystems).  Up to 2 million cells were processed per hybridization following 10x recommendations. Hybridizations were set up in 80 uL of hybridization mix with 20 uL of Human or Mouse WTA probes (10x Genomics PN-2000510 or PN-2000718). For experiments with probe mixing 10 uL of each probe set were used per hybridization. Hybridizations were performed at 42C for 16-24h.  After hybridization samples were diluted in Post-Hyb Wash Buffer and measured by AO-PI staining in a LUNA-FX7™ Automated Cell Counter (Logos Biosystems). For each experiment, we pooled an equal number of cells from each hybridization to have an equal contribution per sample. Cell pools were then washed 3 times in Post-Hyb Wash Buffer for 10 min at 42C. After the washes, cells were resuspended in Post-Hyb Resuspension Buffer, filtered through a Miltenyi Biotec 30 um filter and measured with the cell counter to determine the amount needed for the Chromium X run.   For the GEM encapsulation we followed the 10x Genomics protocol and their guidelines on the volume of cells and reagents required per well according to the targeted cell recovery. After loading the Chip Q and running it on the Chromium X, GEMs were recovered and processed as indicated by 10x Genomics. After processing the GEMs, the product is pre-amplified and indexed to construct the sequencing library according t the 10x Genomics Gene Expression Flex User Guide.
description
not collected
geographic location (country and/or sea)
xenograft LXFS cells grown in mice
growth condition
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
tumor
organism part
Homo sapiens
scientific_name
female
sex
NMRI
strain
LXFS human
title
LXFS lung cancer cell
xenograft