Tapestation 2200

Par or AKTi-R cells were plated in complete RPMI medium and the following day, DNA extraction was performed using the DNeasy kit (Qiagen). Prior to processing by whole exome sequencing, the concentration and integrity of DNA samples was determined using NanoDrop 8000 (Thermo Fisher Scientific) and 2200 TapeStation (Agilent Technologies), respectively. Exome capture was performed using 0.5 μg of genomic DNA and SureSelectXT Human All Exon v5 kit (50 megabases [Mb]) according to manufacturer’s protocol (Agilent Technologies, CA). Fragment size distribution of post-capture amplified libraries was determined with 2200 TapeStation using high sensitivity D1000 screen tape (Agilent Technologies, CA). Concentration of the libraries was measured by Qubit (Thermo Fisher Scientific). Exome capture libraries were sequenced on HiSeq 2500 (Illumina, CA) to generate 75 million paired-end 75 base pair reads. High quality exome-seq reads were mapped to NCBI GRCh38 using GSNAP. Somatic SNVs and INDELs were called by comparing the treatment resistant clones against the parental clones using LoFreq with its default setting. Highly-confident variants were annotated using Ensembl Variant Effect Predictor and filtered with dbSNP 138, ExAC 0.3.1 and RepeatMasker 4.0.5. The functional consequences of somatic variants were annotated using SIFT, PolyPhen and Condel.

Total RNA was extracted from cells using the Qiagen All Prep nucleic isolation kit (Qiagen, Inc.; catalog number 80204, Valencia, CA) as per the manufacturer’s protocol, including the on-column DNase digestion. Quality control of samples was done to determine RNA quantity and quality before their processing by RNA sequencing (RNA-seq). The concentration and the integrity of total RNA samples were determined using NanoDrop 8000 (ThermoFisher Scientific, Waltham, MA) and 2200 TapeStation (Agilent Technologies, Santa Clara, CA), respectively. One microgram of total RNA was used as an input material for library preparation using TruSeq RNA Sample Preparation Kit v2 (Illumina, San Diego, CA). Library size was confirmed using the 2200 TapeStation and high-sensitivity D1K screen tape (Agilent Technologies, Santa Clara, CA), and their concentration was determined by quantitative PCR-based method using Library quantification kit (Kapa Biosystems, Wilmington, MA). The libraries were multiplexed and then sequenced on HiSeq2500 (Illumina, San Diego, CA) to generate 30 M of single-end 50 base pair reads.

Total RNA was extracted using a RNeasy Mini Kit (Qiagen), quantified in a NanoDrop 8000 (ThermoFisher), and assessed for integrity using both 2100 5 Bioanalyzer and 2200 TapeStation (Agilent). Libraries were prepared from 1 μg of total RNA with TruSeq RNA Sample Preparation Kit v2 (Illumina). Library size was confirmed using 2200 TapeStation and High Sensitivity D1K screen tape (Agilent), and concentration was determined by Library quantification kit (KAPA). Libraries were multiplexed five per lane and sequenced in a HiSeq2500 (Illumina) to generate 50 million paired end 75 bp reads. Filtering of fastq sequence files removed poor quality reads (read length < 18 or > 30% of cycles with Phred score < 23). Raw FASTQ reads were aligned to the mouse reference genome (GRCm38-mm10) using GSNAP (with parameters -M 2 -n 10 -B 2 -i 1 -N 1 -w 200000 -E 1—pairmax-rna = 200000—clip-overlap). Reads were filtered to include only the uniquely mapped reads. Differential expression analysis was performed using the voom/limma R package [36 (link)]. Genes were considered differentially expressed if the log2 fold change was > 1 or < -1, and adjusted p-value < 0.05. Pathway analysis was performed with the R package EGSEA [37 (link)].