GSM7734656: mouse RT, R26-SHH, R26P87; Mus musculus; RNA-Seq
Tumor samples cut in small pieces then dissociated 30 min at 37°C in CO2-independent medium (Gibco) + 0,4 g/l of human albumin (Vialebex) with Liberase TL (Roche) 150 mg/ml and DNase 1 (Sigma) 150 mcg/ml. Dissociated cells were then filtered with a 40 mm cell strainer, then washed and resuspended with C02-independent medium + 0,4 g/l of human albumin. Cells were then continuously maintained on ice or at 4°C. In case of lot of blood cells, the Debris removal kit (Miltenyi Biotec) was used according to the manufacturer's protocol. To enrich in tumoral cells (human samples) the Tumor Cell Isolation Kit (Miltenyi Biotec) was used according to the manufactu  rer's protocol. Cells were then resuspended in PBS + BSA 0.04%. Samples were prepared for concentration of 800 cell/mcl. Sample preparations were loaded on a 10x Chromium instrument (10x Genomics) and libraries were prepared using a Single Cell 3' Reagent Kit (V2 chemistry, 10X Genomics) according to the manufacturer's protocol, targeting 1000 recovered cells per sample. 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 Dynabead MyOne Silane Beads (Thermo Fisher Scientific), then amplified with the following protocol: 98°C- 3min; 12x (98°C-15s, 67°C-20s, 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 selects beads. Library and quality assessment were achieved using dsDNA High Sensitivity Assay Kit and Bioanalyzer Agilent System. Indexed libraries were tested for quality, equimolarly pooled and sequenced on an Illumina HiSeq2500 using paired-end 26x98 bp as sequencing mode, targeting at least 50 000 reads par cell.