GSM7594320: Lymph node tissue of patient 7; Homo sapiens; RNA-Seq
Immediately after surgical excision to remove the tumor, we collected fresh samples and placed them in an ice solution containing Hank's balanced salt solution and 1% antifungal antibody, and transferred the tissue into a 4 ℃refrigerator for storage within 20 minutes. Subsequently, a small soft tissue sample was taken and cut into 1 mm3 pieces, washed well using DPBS (cat. no. C14190500BT; Thermo Fisher Scientific, Inc.), and digested by adding collagenase 2 (1 mg/mL) for 45 min at 37 ℃. The cells were filtered using a 100 uM cell filter, and the obtained cell suspension was centrifuged for 5 min at 300 g. The supernatant was removed, and red blood cells were removed using 1× red blood cell lysis buffer (10× diluted to 1×; cat. no. B250015; BioLegend, Inc.). Finally, after washing the cells with PBS, the cells were resuspended using BPBS. Meanwhile, cell viability will be assessed using 0.4% Trypan blue staining. Paracancerous tissues contain more hard bone composition, which presents great challenges in single-cell sequencing. To overcome this limitation, in this study, we first used αMEM (SH30265.01, HyClone) to clean the hard bone three times and bite them into small pieces of about 1-2 mm in diameter. 12 ml of collagenase type II at a concentration of 2 mg/ml (Cat: A004174-0001, Sangon Biotech) was mixed with αMEM containing 100 U/ml penicillin and 100 μg/ml streptomycin (Cat:15140-122, Gibco) in a 50 ml conical tube. The bone snap was then placed in it and digested at 37 °C for 25 min with gentle agitation. Afterwards, the collagenase solution was removed aseptically and the bone pieces were rinsed three times in 10 ml of PBS. After five rounds of digestion, the collagenase solutions from the last two rounds of digestion were combined and the solutions were filtered through a 40 μm filter. Finally, Red Blood Cell Lysis Solution (10×) was used to remove red blood cells according to the manufacturer's instructions. The final single cells from each sample were loaded on a 10× Genomics Chromium Single-Cell Chip along with the single cell master mix and single cell 3' gel beads (10× Genomics) to generate single-cell gel bead-in-emulsions (GEMs). According to the manufacturer's instructions the samples were processed by 10× Genomics V3 barcoding chemistry kits. mRNA in drops were undergo the reverse transcription reactions and then the cDNA amplification was followed by the instructions. And the single cell libraries were sequenced on an Illumina NovaSeq 6000 instrument.