title: GSM4566203: hPBMCs_mice_GM-CSFpos; Homo sapiens; RNA-Seq
library_construction_protocol: PDX tumors were collected in CO2 independent medium (Gibco) within few hours after resection. Tissue were cut into small fragments and submitted to enzymatic digestion using 0.1 mg/ml of Liberase TL (Roche) and 0.1 mg/ml of DNase (Roche) for 30 min. Cells were filtered on 40-μm cell strainer (BD), washed using CO2 independent medium (Gibco) containing 0.4g/ml of human albumin and resuspended for cell counting. Cells were further washed in PBS and stained with specific mAb and submitted to FACS-sorting. After sorting, cells were collected in PBS+0.04% BSA and counted before being submitted to 10x Genomics single-cell RNAseq protocol using 3'Reagents Kit as the following: Single cells were included and barcoded into droplets together with gel beads coated with unique barcodes, unique molecular identifiers (UMI), and poly(dT) sequences, followed by in droplet reverse transcription to generate barcoded full-length cDNA. cDNA was subsequently recovered from droplets, then cleaned up with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific), then amplified with the following protocol: 98 C - 3 min; 12x (98 C - 15 s, 67 C - 20 s, 72 C - 1 min); held at 4 C. Amplified cDNA product was cleaned up using the SPRI select Reagent Kit (Beckman Coulter). Indexed libraries were constructed following these steps: (1) fragmentation, end repair and A-tailing; (2) size selection with SPRI select beads; (3) adaptor ligation; (4) post-ligation cleanup with SPRI select beads; (5) sample index PCR and final cleanup with SPRI select beads. Library quantification and quality assessment were achieved using a dsDNA High Sensitivity Assay Kit and Bioanalyzer Agilent 2100 System. Indexed libraries were tested for quality, equimolarly pooled and sequenced on Illumina Novaseq 6000 S1 using paired-end 26x91bp as sequencing mode, targeting at least 50 000 reads per cell. Libraries were prepared using a Single Cell 30 Reagent Kit (V2 chemistry, 10X Genomics) according to the manufacturer's protocol
attributes.tag: GEO Accession
attributes.value: GSM4566203