GSM6482226: ALKR1275Q SAP development D25-32, cells, scRNAseq; Homo sapiens; RNA-Seq
Prior to scRNAseq the cells of selected time-points were hashed and pooled together for scRNAseq analysis. The time-points D25-26, D27-28, D29-30 and D31-32 were hashed for the WT cells. For the ALK mutant cells time-points D25-27-29-32 were hashed. After dissociating the cells with Accutase for 25 minutes we prepared a 50μl single cell suspension at a concentration between 1x105 cells/mL and 2x107 cells/mL for each hasing group in Cell Staining Buffer (BioLegend, Cat nr. 420201) for cell labelling. 5μl Of cell solution was used to record total cell count and cell viability using Trypan Blue. To the remaining 45μl, 0.1X volume (5 μL) of Human TruStain FcX (BioLegend, Cat nr. 422301) was added. This was allowed to incubate for 10 minutes at 4°C. During this incubation time a solution of 1μg /50μl was prepared for each hashing antibody (B0251 – B0256, see key resource table). The hashing antibody solution was centrifuged at 14,000g at 4°C for 10 minutes. The antibody solution was thereafter carefully withdrawn, whilst avoiding touching the bottom of the tube, and transferred on the corresponding cell solution. This was allowed to incubate for 30 minutes at 4°C, simultaneously the cell staining buffer was cooled down to 4°C. After incubation the cell solution was diluted 10 times by adding 0.9 mL cell staining buffer and transferred to a 12 x 75mm Falcon Round-Bottom Polystyrene Tube. Subsequently we added 2.5ml of cell staining buffer for a final dilution of 35 times. The cell solutions where thereafter  centrifuged at 400g at 4°C for 5 minutes. Next the supernatant was aspirated leaving 50-100μl, this was repeated two more times for a total of 3 washes. It is very important to perform these washes carefully, leaving as little supernatant as possible. After the last wash the different cell solutions (each with their own hashtag) were combined into one tube, centrifuged and resupsended in DMEM-F12, as this medium was compatible with the 10XGenomics pipeline, for a final concentration of 1000 cells/μl. The single-cell RNA-Seq was performed on Chromium instrument (10X genomics) following the user guide manual (Reagent Kit 3' v3.1). Each sample, containing approximately 10,000 cells at a final dilution of ~1,000 cells/µl was loaded onto the cartridge following the manual. The viability of cells was above 80%, as confirmed with 0.2% (w/v) Trypan Blue staining (Countess II). The individual transcriptomes of encapsulated cells were barcoded during RT step and resulting cDNA purified with DynaBeads followed by amplification per manual guidelines. Next, PCR-amplified product was fragmented, A-tailed, purified with 1.2X SPRI beads, ligated to the sequencing adapters and indexed by PCR. The indexed DNA libraries were double-size purified (0.6–0.8X) with SPRI beads and sequenced on Illumina NovaSeq S4 platform (R1 – 26 cycles, i7 – 8 cycles, R2 – 70 cycles or higher).