Illumina NovaSeq 6000 paired end sequencing; High complexity cellular barcoding and clonal tracing reveals stochastic and deterministic parameters of radiation resistance
Cells suspensions for RNA extraction was obtained from exponentially growing cultures by mild enzymatic detachment using a 0.05% trypsin / 0.02% EDTA solution in phosphate buffered saline (PBS, PAN Biotech):  FaDu (ATCC® HTB-43™) was cultured in Minimum Essential Medium (MEM, Gibco™/Life Technologies) with 10% Fetal Bovine Serum (FBS, Sigma-Aldrich) and 1% Penicillin and Streptomycin (10,000U/mL / 10mg/mL; Gibco™/Life Technologies).  Patient-derived cell lines were cultured in Dulbeccos´s Modified Eagle Medium (DMEM, Biochrom) with 10% FBS and 1% Penicillin and Streptomycin.  Cells were cultured at 37°C in a humidified environment and 5% CO2 atmosphere. Cultures were regularly tested to be free from mycoplasma using Venor®GeM Classic Kit (Minerva Biolabs).  Cells were passaged by reaching 90% confluency using Trypsin/EDTA.  Total RNA was extracted using Quiagen AllPrep DNA/RNA Mini Kit according to manufacturer's protocol. In brief, 5x106 cells were disrupted by adding 600 µl buffer RLT Plus and subsequent vortexing. For homogenization, the lysate was passed 5 times through a 20-gauge needle fitted to an RNase-free syringe. The lysate was transferred to an AllPrep DNA spin column and centrifuged. The gDNA bound column was stored at 4°C for later DNA purification. For RNA extraction 600µl 70% ethanol was added to the RNA-containing flow-through and transferred to an RNeasy spin column. After washing, total RNA was eluted from the column membrane by adding 50µl nuclease-free water. RNA concentrations were determined using Nanodrop spectrophotometer and RNA was stored at -80°C. RNA sequencing was performed on FaDu, HNO407 and HNO206 (3 biological replicates). 1.2µg high purity total RNA (defined as having an RNA integrity number (RIN) greater than 8.0) was used as input for RNA seq library construction. Library preparation for RNA-seq was performed using the Illumina TruSeq® Stranded mRNA Library Preparation Kit, following manufactures recommended standard protocols.