Smart-seq3 Protocol v3

It is absolute fine to use standard desalting instead of HPLC, both works fine in our hands, same goes for hand-mixed vs machine mixed degenerate bases.Using the regular DNA oligos service at IDT should provide based on their QC full length oligos.• We use custom Nextera Indexes primers (standard 25 nmol oligo preps from IDT, delivered at 200 nM concentration in IDTE buffer) and we typically get performance that is indistinguishable from Illumina's primers.For making your own primers, we recommend using the "DNABarcodes" R package.using the following settings: Barcode length: 10 bp (or 8bp like Illumina primers, depending on the amount of cells you need indexed and sequenced at the same time) Minimal levenshtein distance: 3 Filter out homopolymers >= 3 Filter for uneven GC content Additionally, there seems to be an artifact on the NovaSeq platform for i5 index primers starting with the bases "AC", so we recommend to avoid those too!(see supplementary information in this paper: https://bmcgenomics.biomedcentral.com/articles/10.