GSM9764198: Parse Evercode WT scRNA-seq — human tumor cell, shLOX KD rep 2 (shLOX-T-2); Homo sapiens; RNA-Seq
Tumors were excised at week 7 post-implantation, minced with blades, and enzymatically dissociated in 5 mL Miltenyi Human Tumor Dissociation Kit reagent (Cat. 130-095-929) per tumor in a 50 mL tube at 37 C with continuous rotation for 50 min. Undissolved tissue was removed with a 70 um cell strainer rinsed with 20 mL DMEM. The suspension was centrifuged (300 x g, 7 min), aspirated, reconstituted in DMEM, and passed through a 30 um strainer to remove aggregates. Dead cells were depleted with the Miltenyi Dead Cell Removal Kit (Cat. 130-090-101) using 100 uL microbeads per 1e7 cells, 15 min RT, then column separation on a MACS magnet; the live-cell effluent was collected. CD11b+ immune cells were then separated from CD11b- tumor cells using Miltenyi CD11b MicroBeads UltraPure, mouse (Cat. 130-126-725). Both fractions (CD11b- tumor + CD11b+ immune) were fixed using the Parse Biosciences cell-fixation kit per manufacturer's instructions, yielding 12 fixed-cell suspensions (6 tumors x 2 fractions). Single-cell transcriptome libraries were prepared with the Parse Biosciences Evercode WT v2 kit per the manufacturer's instructions. The 12 fixed-cell suspensions were combinatorially barcoded together using 2 Round-1 (BC1) wells per sample — tumor samples in row C (C1:C12, alternating pLKO / shLOX) and immune samples in row D (D1:D12, alternating pLKO / shLOX) of the 48-well Round-1 plate. Barcoded cells were pooled and split into 8 sublibraries of approximately 12,500 cells each (S1_WT_A1 ... S8_WT_H1). Final libraries were QC'd by Qubit fluorometry and Bioanalyzer. Sublibraries were sequenced on an Illumina NovaSeq X Plus (PE150) at Novogene (Davis); 4 of 8 sublibraries (S1/S2/S3/S5) were re-sequenced on a second flowcell for additional depth.