GSM8085952: Immunocompetent_CellplexOligo_Library2; Mus musculus; RNA-Seq
Mice implanted with scaffolds were sacrificed on day 14 post implantation and the scaffolds with tumor microenvironment were isolated aseptically for single-cell RNA sequencing experiments. One half of the collected scaffolds from each condition was minced into 1-2 mm pieces and transferred to 1.5 ml Eppendorf tube for dissociation into a single cell suspension. Tissue pieces were then digested in a tissue dissociation media with 5mg/mL dispase, 2.5 mg/mL collagenase I, and 2.5 mg/mL collagenase II in 1x basal culture medium in a 37°C incubator. At the end of the digestion, the cells were passed through a 40 µm filter and centrifuged into a pellet. To remove erythrocytes from the suspension, samples were resuspended in an ammonium-chloride-potassium (ACK) lysis buffer for 3-5 minutes and centrifuged at 180g for 6 minutes. For immunocompromised mice, samples were then washed 3x in PBS with 0.04% BSA and then resuspended at 1 × 106 cells per ml. Cells from each sample were stained with Trypan Blue and cell viability was calculated on an automated cell counter (Countess II) before loading the cells on 10x Chromium. We used these cell viabilities to adjust the number of cells loaded on 10x Chromium to get the desired number of transcriptomes from viable cells for each sample (5000 cells per sample). For immunocompetent mice, samples were washed twice in PBS with 0.04% BSA and then resuspended at 0.5 × 106 cells in 1 ml total buffer. The cells were then centrifuged and the supernatant was removed without disturbing the pellet. Distinct 10x Genomics Cell Multiplexing Oligos were then added to all samples and incubated for 5 minutes at room temperature. Samples Control-M-Bone, Control-DM-Bone, Tumor-M-Bone, Tumor-DM-Bone were tagged with CMO301, CMO302, CMO303, and CMO304 respectively. Labeled cell suspensions were then washed 3x in pre-chilled wash and resuspension buffer containing 1% BSA in 1x PBS as recommended in the manufacturer's Cell Multiplexing Oligo Labeling protocol for Single-cell RNA Sequencing. The cells were resuspended in the resuspension buffer at 1500 cells/ul and were counted on an automated cell counter (Countess II). Labeled cells from individual samples were pooled in equal proportions, stained with Trypan Blue, and cell viability was calculated on an automated cell counter (Countess II) before loading the cells on 10x Chromium. We used these cell viabilities to adjust the number of cells loaded on 10x Chromium to get the desired number of transcriptomes from viable cells for each sample (5000 cells per sample; 20,000 total cells for four samples). For immunocompromised mice, single-cell suspensions for individual samples were directly loaded on the Chromium platform (10x Genomics) with one microfluidic channel per condition and ~5000 target cells per channel. Single-cell mRNA libraries were made using the Chromium Next GEM Single Cell 3' Library Construction V3.1 Kit according to the manufacturer's protocol. Sequencing Libraries sequenced on an Illumina NextSeq 500/550 using 75 cycle high output kits (Index 1 = 8bp, Index2 =8bp, Read 1 = 28, and Read 2 = 55bp). Raw sequencing data was aligned to a combined human and mouse genome reference (assembly: GRCh38 and mm10) using the Cell Ranger 6.1.1 pipeline (10x Genomics) to obtain single-cell gene expression matrices for individual samples. For immunocompetent mice, oligo-tagged and pooled single-cell suspension derived from all four experiment conditions was loaded on a single microfluidic channel of the Chromium platform (10x Genomics) with a target of ~16,000 cells (~4,000 cells per experiment condition). A single-cell mRNA library and a cell multiplexing oligo library were made using the Chromium Next GEM Single Cell 3' Library Construction V3.1 Kit with Feature Barcode technology for Cell Multiplexing, according to the manufacturer's protocol. Sequencing Libraries sequenced on an Illumina NextSeq 500/550 using 75 cycle high output kits (Index 1 = 10bp, Index2 =10bp, Read 1 = 28, and Read 2 = 90bp). Raw sequencing data was aligned to a combined human and mouse genome reference (assembly: GRCh38 and mm10) and demultiplexed using the Cell Ranger 6.1.1 pipeline (10x Genomics).