PS-PILT-g011
Homo sapiens
Protocols: PC9 cells were grown in RPMI 1640 with Glutamax (Gibco) and 10% FBS. NIH-3T3 cells were grown in DMEM (Gibco) with L-glutamine and 10% FBS. Cells were dissociated using Tryple Express (no phenol red) (Gibco) and made into single-cell suspension, followed by filtration through 30uM Miltenyi pre-separation filters and counting using AO/PI double stain (Nexcelom).  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. Libraries were sent for sequencing to the Harwig Medical Foundation with the Illumina Nova-Seq 6000 or internally at Single-cell Discoveries with the Illumina Nova-Seq X sequencing platform following 10x recommended settings. For the PC9 spiked samples we sequenced at a depth of 15,000 reads. For the experiments with the probe mixing strategies we sequenced at a depth of 30,000 reads per cell.
isolate
not applicable
disease
lung adenocarcinoma
library
PS-PILT-g011
organism
Homo sapiens
organism
Homo sapiens
cell_line
PC-9
collection date
not collected
scientific_name
Homo sapiens
common name
human
cell_type
lung cancer cell
geographic location (country and/or sea)
not collected