title: GSM5655250: scRNA-seq Human Pancreatic Cancer replicate 1; Homo sapiens; RNA-Seq
library_construction_protocol: Tumor Tissue Processing  Tumor tissue specimens from mouse or human specimens was minced on ice. The tissue was then digested for 3x 30 minutes in a 37C water bath agitator in 0.5 mg/mL collagenase (collagenase type IV, ThermoFisher) digest buffer in Medium 199 (HyClone, GE Healthcare) consisting of 5% fetal bovine serum (Gibco FBS, ThermoFisher), DNase I (Worthington), Poloxamer 188 (Cat. P5556-100ML, Sigma), HEPES, and CaCl2. The digest was quenched with quench media (DMEM (Gibco DMEM, ThermoFisher) with 15% FBS), then centrifuged at 300 x G for 5 minutes at 4C, resuspended in quench media, and filtered through 100, 70, and 40m cell strainers (Falcon cell strainer, ThermoFisher). Red blood cell lysis was performed using Hybri-Max (Sigma) per the manufacturers protocol. Cells were counted and re-suspended for further processing. Single Cell Barcoding, Library Preparation, and Sequencing.   Cell suspensions from individual human and mouse samples were labeled with TotalSeq Series B hashtag oligonucleotide-labeled antibodies (BioLegend). Single-cell RNA-seq (scRNA-seq) was then performed at the Stanford Functional Genomics Facility (SFGF) for droplet-based microfluidics using the 10x Chromium Single Cell platform (Single Cell 3' v3, 10x Genomics, USA). Droplets of the cellular suspensions, reverse transcription master mix, and partitioning oil were mixed, loaded onto a single cell chip, and processed on the Chromium Controller. Reverse transcription was performed, and cDNA was amplified using a BioRad C1000 Touch thermocycler, with cDNA size selected using SpriSelect beads (Beckman Coulter, USA). An Agilent Bioanalyzer High Sensitivity DNA chip was used to analyze cDNA for qualitative control purposes; cDNA was then fragmented using the proprietary fragmentation enzyme blend for 5min at 32°C, followed by end repair and A-tailing at 65°C for 30 min. DNA was double-sided size selected using SpriSelect beats. Sequencing adaptors were ligated to the cDNA at 20°C for 15min. cDNA was amplified using a sample-specific index oligo as primer, followed by another round of double-sided size selection using SpriSelect beads. Final libraries were analyzed on an Agilent Bioanalyzer High Sensitivity DNA chip for qualitative control purposes. Libraries were sequenced on a HiSeq 4000 Illumina platform targeting 50,000 reads per cell.