GSM6341016: scRNA-seq_E990 iExo-Myc#1 treated mouse; Homo sapiens; OTHER
Nuclei from frozen tumor were isolated from ~40 mg of both iExo-Myc and iExo-Sca tissue samples using the EZ Prep buffer (Sigma). Nuclei and custom-made barcode (BC) beads (ChemGenes) were loaded at a concentration of 300,000 nuclei/ml in NSB and 350,000 beads/ml in lysis buffer respectively in separate 3 ml syringes. A 75-micron microfluidic device fabricated in-house was used to run the DroNc-Seq experiments at 1.5, 1.5 and 12 ml/hr flow-rated for the nuclei, BC beads and carrier oil channels respectively, with approximately 22 minutes per collection. Following collection, drops were broken, and uniquely barcoded cDNA was generated from the barcoded polyT-mRNA hybrids using reverse transcription. These barcoded cDNA attached to the polymer beads called STAMPs, and washed several times in TE buffer containing tween and SDS, and then treated with exonuclease I enzyme (Sigma) to digest and remove any barcodes that did not have a cDNA attached to it. The number of recovered beads or STAMPS were counted. Approximately 20 wells in a PCR plate at 5000 beads per well, and amplified through 15 cycles of PCR. Supernatant from each well were pooled and cleaned with 0.6x SPRI beads. Purified cDNA product was quantified using a Qubit DNA assay kit and 450-650 pg of each sample was used as input for Nextera library preparation reactions (12 cycles). Tagmented libraries were quantified on Agilent BioAnalyzer High sensitivity chip before sequencing.