GSM8848674: tumor tissue of squamouspapillary craniopharyngioma from case CP9, biological replicate2; Homo sapiens; RNA-Seq
A single-cell suspension of the craniopharyngioma samples was digested via stirring at 37_x000E_C for 90 min using collagenase I 60 (160 mL, 100 mg/ mL) and collagenase VII 61 (400 mL, 100 mg/mL; Worthington, OH, USA) immediately after surgical removal. The suspension was then filtered through a 40-mm cell strainer (Corning, NY, USA) to remove debris and undigested large tissue fragments. After allowing the cells to settle, red blood cells were isolated using ACK lysing buffer (Thermo Fisher Scientific, MA, USA), and dead cells were removed using a dead cell removal kit (Miltenyi Biotec, Bergisch Gladbach, Germany); the resultant cell suspension contained approximately 20,000 cells per sampl The obtained cell suspension was prepared following the specifications of 103 Genomics, using 103 Chromium Next GEM single-cell 3ʹ re- agent kits and the library construction kit (103 Genomics, CA, USA). The cell suspension was loaded into the 103 Genomics Chromium Controller, creating gel beads in emulsion (GEM). After generating the GEM, the suspension was incubated at 53_x000E_C for 45 min in a thermal cycler (Veriti, Applied Biosystems, MA, USA). A barcode-linked polyA cDNA with barcodes at the 5ʹ end was produced via adding template switch oligos linked to cell barcodes and unique molecular identifiers (UMIs). The GEMs were then disrupted, and single-stranded cDNA was cleaned using DynaBeads MyOne Silane Beads (Thermo Fisher Scientific). The cDNA was amplified (98_x000E_C for 3 min, 11 cycles of 98_x000E_C for 15 s, 63_x000E_C for 20 s, and 72_x000E_C for 1 min) and the quality was assessed using Agilent TapeStation (Agilent Technologies, CA, USA). The cDNA was enzymatically fragmented, end-repaired, A-tailed, and subjected to size selection on both ends using SPRIselect beads (Beckman Coulter, CA, USA). It was then ligated with adapters provided in the kit. Sample-specific indices were introduced into each library via 14-cycle poly- merase chain reaction amplification using kit-supplied indices (98_x000E_C for 45 s, 14 cycles of 98_x000E_C for 20 s, 54_x000E_C for 30 s, 72_x000E_C for 20 s, 72_x000E_C for 1 min, and holding at 4_x000E_C). Indexed libraries underwent a second round of size selection. The library was quantified and quality was assessed using Agilent TapeStation. Libraries were clustered on a NovaSeq 6000 paired-end read flow cell (Illumina, CA, USA). Sequencing was per- formed with reads for R1 (containing 103 barcodes and UMIs), I7 index (8 cycles; sample index), and R2 (89 cycles; transcript). The 103 Ge- nomics Cell Ranger Single-Cell Software was used for sample demultiplexing, alignment to the human reference genome (hg19), filtering, UMI counting, single-cell 3ʹ end gene counting, and QC as per manufacturer's instructions.