Miseq reagent kit v2 300 cycle

A total of 35 amplicons from each patient were pooled in equimolar ratios. According to the manufacturer’s protocol, 1 ng of the pooled DNA fragments was subjected to the Nextera XT procedure (Illumina) using transposome technology. Finally, using the Nextera XT DNA Sample Preparation Kit (Illumina) and the Nextera^® XT Index Kit (96) (Illumina), we obtained one hundred and seven both-side indexed DNA libraries ready for high-throughput sequencing. The normalization of all libraries was carried out with magnetic beads, according to the Nextera XT procedure. Sequencing on the Illumina MiSeq platform was performed as paired-end targeted resequencing using the MiSeq Reagent Kit v2 (300 cycle) (Illumina). To verify the detected variants, randomly Sanger sequencing analysis was performed on Applied Biosystems 3500 Genetic Analyzer using BigDye^® Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific).

Viable CRKP isolates (n = 85/86) were subcultured overnight on 2% blood agar in an oxygen-enriched incubator at 37°C. Genomic DNA was extracted using the Quick-DNA Fungal/Bacterial Miniprep Kit (Zymo Research Corp., Irvine, CA, USA) followed by library preparation after DNA quality control using the Illumina DNA Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Library quality control was performed using an Agilent DNA 1000 kit (Agilent Technologies, CA, USA) and Qubit^™ dsDNA HS Assay Kit (Invitrogen, Life Technologies, CA, USA). Each sequencing run contained the PhiX V3 internal sequencing control (Illumina) spiked at 2%. Sequencing was performed on an Illumina^® MiSeq^™ instrument using a MiSeq^™ Reagent Kit v2 (300-cycle) (Illumina) as per the manufacturer’s instructions.

For human genome cleavage assays, 2.0 × 10⁴ HEK293FT cells in 96-well plates were cotransfected with combinations of Fz expression plasmid (80 ng) and ωRNA expression plasmid (20 ng). After 3 days of incubation at 37 °C, the supernatant was removed and cells were resuspended in 40 µl QuickExtract DNA extraction solution (Lucigen, QE09050) and cycled at 65 °C for 15 min, 68 °C for 15 min, then 95 °C for 10 min to lyse cells. Two microlitres of lysate were used as the template for each 12.5 µl PCR reaction. Target sites were amplified with NEBNext High-Fidelity 2X PCR Master Mix (NEB, M0541L) under the following thermal cycling conditions: one cycle, 98 °C, 30 s; 15 cycles, 98 °C, 10 s, 65 °C, 20 s, 72 °C, 30 s; one cycle, 72 °C, 30 s; 4 °C hold. One microlitre of this first PCR product was used for the template for each 10-µl second PCR reaction: one cycle, 98 °C, 30 s; 15 cycles, 98 °C, 10 s, 63 °C, 20 s, 72 °C, 30 s; one cycle, 72 °C, 30 s; 4 °C hold (total 30 cycles for first and second PCR reactions). Amplicons were sequenced using a MiSeq Reagent Kit v.2, 300-cycle (Illumina, MS-102-2002). Indel efficiency was quantified using the established CRISPResso2 v.2.0.20b pipeline^{34 (link)}.

Initially, libraries of the technical testing cohort were prepared with the TruSeq Amplicon Cancer Panel (TSACP), covering 225 amplicons of 48 cancer associated genes, following exactly the manufacturer’s protocol (Illumina, San Diego, USA). Since this did not yield reliable libraries for the different types of FFPE tissue specimens (Additional file 1: Table S1A), modifications to the library protocol were made to the first hybridization step with oligo pools and to subsequent PCR cycling parameters (see Additional file 1: Table S1B). The comparison of the performance of these different library protocols is presented for the technical testing and validation cohorts (Additional file 1: Figure S1).
The selection of specific library protocols according to DNA amount and quality for the 70 DNA samples of the CRC study cohort is provided together with the tumour cell content in Additional file 1: Table S2).
Libraries were examined for quantity and quality using the Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, USA) and subjected to clean up, normalization and pooling according to the manufacturer (Illumina, San Diego, USA). MiSeq sequencing runs were performed with each 16–20 pooled libraries using the MiSeq Reagent Kit v2 (300 cycle) (Illumina, San Diego, USA) and paired-end sequencing with 2 × 151 bp.

cDNA from single cells was obtained using a modified version of the SmartSeq2 protocol^{81 (link)}. Briefly, single cells were sorted into plates containing lysis buffer and cDNA was generated by template switch reverse transcription using SMARTScribe Reverve Transcriptase (Clontech, catalog: 639538), a template-switch oligo and primers kit designed for the constant regions of Trac and Trbc genes. TCR amplification was achieved by performing two rounds of nested PCR using Phusion High-Fidelity PCR Master Mix (New England Biolabs, catalog: M0531S). During the first PCR priming, indexes were included to identify each cell. A final PCR was performed to add the Illumina adapters. TCR libraries were sequenced on Illumina MiSeq using MiSeq Reagent Kit V2 300-cycle (Illumina). FASTQ files were demultiplexed for each cell. Sequences from clones were analysed using MiXCR^{82 (link)}. Post-analysis was performed using VDJtools^{83 (link)}. The CDR3 and TRBV usage data generated in this study have been deposited in the Sequence Read Archive under accession code PRJNA1075074.