GSM9038579: Lung metastasis, MDA-MB-231 xenograft model, Mouse 43975; Homo sapiens; RNA-Seq
Post-euthanasia, organs were harvested, washed in cold 1× PBS to remove blood, and either processed immediately or minced into small fragments and cryopreserved in 5% DMSO-FBS in liquid nitrogen. For immediate processing, tissues were finely minced using stacked blades in 100–200 µL FV media until a homogenous paste was obtained, then transferred to 15 mL Falcon tubes containing 4.5 mL FV media (5% FBS added fresh) and kept on ice.  Enzymatic dissociation was initiated with 500 µL Collagenase-Hyaluronidase (C-H) per tube, incubated at 37°C on a rotating wheel for 40 minutes. At 15–20 minutes, tissues were mechanically disrupted using 5 mL serological pipettes. Dissociation efficiency was assessed using a 96-well plate, and tubes were returned to the wheel for another 15–20 minutes. After final pipetting, reactions were stopped by placing tubes on ice and adding cold FACS buffer (2% FBS in PBS), followed by centrifugation at 1300 rpm for 6 minutes.  Red blood cell lysis was performed using 1.5 mL freshly prepared RBL buffer (1:10 in Buxter water), incubated for 2 minutes, then stopped with FACS buffer and centrifuged. Pellets were resuspended in 10 mL FACS buffer with 50 µL DNase and filtered through pre-wetted 40 µm strainers to collect single-cell suspension 1 (SCS1).  Filters were inverted and flushed with 12 mL FACS media to recover retained tissue, centrifuged, resuspended in 100 µL DNase, and treated with 1–2 mL TEG trypsin at 37°C for up to 2 minutes using a cut P1000 tip. Trypsin was neutralized with FACS buffer, and cells were centrifuged and resuspended in 10 mL FACS buffer + 100 µL DNase, then filtered through activated 40 µm strainers to yield SCS2.  Both SCS1 and SCS2 were centrifuged (1300 rpm, 6 minutes), combined in 2 mL FACS buffer, and a 50–100 µL aliquot was reserved as an unstained control. Remaining cells were pelleted and stained with 1 mL of pre-titrated antibody solution, incubated at 4°C for 40 minutes with gentle shaking, washed twice, and resuspended in 500 µL FACS buffer for sorting. GFP-expressing MDA-MB-231 cells were sorted and processed for scRNA-seq following 10x Genomics protocols. Single-cell suspensions were processed using the Chromium Controller (10x Genomics) and the Single Cell 3′ Reagent Kits v2 (PN-120237), following the manufacturer's instructions (10x Genomics, CG00052, Rev F). Cells were encapsulated into nanoliter-scale Gel Bead-in-Emulsions (GEMs), where cell lysis and reverse transcription occurred, barcoding each cDNA molecule with a unique 10x barcode and UMI. Following GEM-RT incubation, emulsions were broken and cDNA was purified using Silane Dynabeads. Amplified cDNA was enzymatically fragmented and processed through end repair, A-tailing, adaptor ligation, and indexing PCR using sample-specific barcodes (Chromium i7 Sample Index Plate, PN-220103). Libraries were quality controlled using Bioanalyzer or TapeStation, quantified, and sequenced on an Illumina platform using a paired-end 2×150 bp configuration, with Read 1 containing 26 bp (barcode + UMI), i7 index of 8 bp, and Read 2 of 98 bp.