Unique dual index barcodes

DNA library preparation of micro-dissected tissue samples was undertaken using a bespoke low-input enzymatic-fragmentation-based library preparation method^{2 (link)–4 (link),37 (link)}. This method was employed as it allows for high quality DNA library preparation from a very low starting quantity of material (from 100-500 cells). In brief, gDNA was purified from cell lysates using bead purification. Enzymatic fragmentation, end-repair and dA-tailing was performed using NEBNext Ultra II FS DNA Library Prep Kit (New England BioLabs). Indexing and PCR amplification was subsequently performed (12 cycles). DNA library concentration was assessed after library preparation and used to guide choice of samples to take forward to DNA sequencing. The minimum library concentration was 5 ng/µL and libraries with >15 ng/µL were preferentially chosen. 150 bp paired-end Illumina reads were prepared with Unique Dual Index barcodes (Illumina). DNA sequencing was undertaken on a NovaSeq 6000 platform using an XP kit (Illumina). Samples were multiplexed in pools of 6-24 samples. Pools were sequenced to achieve a coverage of ~30x per sample.

DNA library preparation of microdissected tissue samples was undertaken as previously described, using a bespoke low-input enzymatic-fragmentation-based library preparation method^{28 (link),29 (link),31 (link),66} . This method was employed as it allows for high-quality DNA library preparation from a very low starting quantity of material (100–500 cells). DNA library concentration was assessed after library preparation and used to guide the choice of samples to take forward to DNA sequencing. Minimum library concentration was 5 ng µl^–1, and libraries with >15 ng µl^–1 were preferentially chosen; 150-bp, paired-end Illumina reads were prepared with Unique Dual Index barcodes (Illumina).
DNA sequencing was undertaken on a NovaSeq 6000 platform using the XP kit (Illumina). Samples were multiplexed in pools of 6–24, then sequenced to achieve a coverage of ≥30×.