title: GSM6514308: Treated, replicate 3, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Metastases from tumor-bearing mice were microsurgically dissected using sterile micro-surgical tools. SS, IF and F regions were dissected in negative controls. Later, samples were disrupted into single cell suspension as described for flow cytometry, using sterile nuclease-free tools. Single cells were barcoded using the 10x Chromium Single Cell platform, and cDNA libraries were prepared according to the manufacturer's protocol (Single Cell 3′ v3, 10x Genomics, USA). In brief, cell suspensions, reverse transcription master mix and partitioning oil were loaded on a single cell chip, then run on the Chromium Controller. Reverse Transcription was performed within the droplets at 53 °C for 45 minutes. cDNA was amplified for a 12 cycles total on a Biometra thermocycler. cDNA size selection was performed using SpriSelect beads (Beckman Coulter, USA) and a ratio of SpriSelect reagent volume to sample volume of 0.6. cDNA was analyzed on an Agilent Bioanalyzer High Sensitivity DNA chip for qualitative control purposes. cDNA was fragmented using the proprietary fragmentation enzyme blend for 5 minutes at 32 °C, followed by end repair and A-tailing at 65 °C for 30 minutes. cDNA was double-sided size selected using SpriSelect beads. Sequencing adaptors were ligated to the cDNA at 20 °C for 15 minutes. cDNA was amplified using a sample-specific index oligo as a 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 quality control. cDNA libraries were sequenced on a  281 NextSeq500 Illumina platform aiming for 50,000 reads per cell. Base calls were converted to reads with the software Cell Ranger (10x Genomics; version 3.1) Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.