title: GSM5743023: Papillary thyroid cancer from left lobe in patient 2; Homo sapiens; RNA-Seq
library_construction_protocol: All freshly resected thyroid specimens were immediately washed twice  with phosphate-buffered saline (PBS) and digested with digestive enzyme mixture containing 10 ml pre-warmed RPMI 1640 (ThermoFisher Scientific), 1 mg/ml type IV collagenase (Sigma), 2 mg/ml dispase (Roche), and 10 U/µl DNase (Roche) for an hour at 37°C. The reaction was deactivated by adding 10% FBS (ThermoFisher Scientific). Cell suspensions were filtered using a 70 μm filter and then centrifuged at 500 rpm for 6 min at 4°C to pellet dead cells and red blood cells. The cells were washed twice and suspended in PBS with 0.5% bovine serum albumin (BSA, Sigma). Cell suspension (300-600 living cells per microliter determined by Count Star) was loaded onto the Chromium single cell controller (10x Genomics) to generate single-cell gel beads in the emulsion using single cell 3 'Library and Gel Bead Kit V3.1(10x Genomics, 1000121) and Chromium Single Cell G Chip Kit (10x Genomics, 1000120) according to the manufacturer's protocol. In short, single cells were suspended in PBS containing 0.04% BSA.   About 6,000 cells were added to each channel, and the target cell that will be recovered was estimated to be about 3,000 cells. Captured cells were lysed and the released RNA were barcoded through reverse transcription in individual GEMs. Reverse transcription was performed on a S1000TM Touch Thermal Cycler (Bio Rad) at 53°C for 45 min, followed by 85°C for 5 min, and hold at 4°C. The cDNA was generated and then amplified, and quality assessed using an Agilent 4200 (performed by CapitalBio Technology, Beijing). According to the manufacture's introduction, Single-cell RNA-seq libraries were   constructed using Single Cell 3' Library and Gel Bead Kit V3.1. The libraries were finally sequenced using an Illumina Novaseq6000 sequencer with a sequencing depth of at least 100,000 reads per cell with pair-end 150 bp strategy (performed by CapitalBio Technology, Beijing).