GSM6208743: WB1P-Myc sample 1 miseq; Mus musculus; RNA-Seq
Single cell suspensions of the tumors were sorted by FACS to isolate live single cells (FSC-A x FSC-H to define singlets, dapi-negative cells to define live cells). After sorting the cells were subjected to Dropseq RNA sequencing according to the protocol of Macosko et al.[61] using WB1P cells (275 cells/ul) and WB1P-Myc cells (270 cells/ul) both in 1xPBS+0.01%BSA. Dropseq beads 'MACOSKO-2011-10' were purchased from ChemGenes. Ready-made Drop-Seq microfluidic devices were purchased from Nanoshift. During droplet generation, of both samples multiple 5 minute fractions of droplets were collected and based on droplet quality assessment for each sample 4 fractions were selected for further processing according to “Drop-seq laboratory protocol v3.1”. After breakage, reverse transcription and Exocuclease I treatment, of each fraction the cDNA was amplified in triplicate PCR reactions. After PCR the amplified cDNA products were pooled, cleaned by a 0.6X AMPure XP bead cleanup and quantified on a 2100 Bioanalyzer Instrument following the manufacturer's protocol “Agilent High Sensitivity DNA Kit” (G2938-90321, Agilent Technologies). This procedure was done twice for every fraction selected. The amplified cDNA product was concentrated by speedvac and all was used as input for the final Nextera XT library preparations. All Libraries were quantified on a 2100 Bioanalyzer Instrument following the manufacturer's protocol “Agilent High Sensitivity DNA Kit” and diluted to 10 nM before paired-end sequencing on the MiSeq and HiSeq2500 Illumina sequencing platforms. Sequencing was performed using 25 cycles for Read 1, 8 cycles for Read i7 and 117 cycles for Read 2, using MiSeq Reagent Kit v3 (150-cycle) (MS-102-3001, Illumina), HiSeq PE Rapid Cluster Kit v2 (PE-402-4002, Ilumina) and HiSeq Rapid SBS Kit v2 (200 cycles) (FC-402-4021, Illumina). Sequencing was performed on the MiSeq and HiSeq2500 Illumina sequencing platforms.