Biomek fxp automation

A small piece of each snap-frozen primary tumor was pulverized using the T-prep method (Covaris, cat # 520097) on dry ice, followed by implementation of Agencourt RNAadvance (Beckman Coulter, cat # A32645) on a Biomek® FXP automation workstation (Beckman Coulter) to isolate RNA. We used 150 ng RNA to prepare libraries with TruSeq Stranded Total RNA kits (Illumina, cat #RS-122–2301). From FFPE tissues, we deparaffinized four 10-μm sections, rehydrated these in an ethanol/water series, and extracted RNA using AllPrep DNA/RNA FFPE kits (Qiagen). We measured concentrations using the Quant-iT RiboGreen RNA assay (Thermo Fisher) and assessed quality on an Agilent 2100 Bioanalyzer using the Agilent RNA 6000 Nano kit. Ribosomal and mitochondrial RNAs were removed using biotinylated, target-specific oligonucleotides combined with Ribo-Zero rRNA removal beads and the TruSeq Stranded Total RNA kit (Illumina). 75-bp single-end reads were sequenced on a NextSeq 500 Instrument (Illumina).

GUIDE-seq library preparation was performed as previously described^{12 (link)}. Briefly, genomic DNA was purified with Agencourt DNAdvance using a BioMek Fx^P automation system (both from Beckman Coulter). Genomic DNA was sheared to average fragment size of 500 bp by Covaris E220 sonication (Covaris) and purified with SPRI magnetic beads. Genomic DNA was quantified by Qubit (Invitrogen), and 400 ng was used for GUIDE-seq library preparation. Genomic DNA was treated with End-repair Mix (Qiagen) and A-tailed with Taq polymerase (Fisher), ligated to single-tailed sequencing adapters and purified with SPRI magnetic beads. The adapter-ligated library was subjected to two rounds of nested PCR using dsODN sense- and antisense-specific primers in separate reactions for each sample, and then purified with SPRI magnetic beads. Libraries were quantified with Kapa qPCR Library Quantification kit (Kapa). Equimolar amounts of samples were pooled for GUIDE-seq libraries were sequenced with 150 bp paired end reads on Illumina NextSeq 550 or HiSeq 2500 sequencers. GUIDE-seq analysis was performed as previous described^{12 (link)}.