Phusion hot start flex

Primers (Integrated DNA Technologies, Heverlee, Belgium) were designed by hand (aided by IDT OligoAnalyzer for Tm values) with complementary regions of 30 bases for hybridization. Delta G-values for these overhangs spanned from 35.2 to 51 Kcal/mol (28 (link)) (calculated using default settings). One primer per amplification reaction had a biotinylated 5′-end and 5 pmol of each primer was used in a 50 μl PCR reaction. DNA parts were amplified with Phusion^® Hot-Start Flex (2 U/μl, New England Biolabs, Ipswich, MA, USA) by the following PCR program: 98°C for 30 s, 30 cycles of 98°C 8 s, 25 s annealing with temperature depending on primer melting temperature and 72°C for 20 s/kb, before ending with 72°C for 7 min followed by 4°C hold. Sequences are detailed in the supplementary information.

Approximately one million beads (0.1 VPB) were transferred to a 50 μl PCR mixture containing 1x Phusion Hot Start Flex (New England BioLabs; Cat. No. M0535), 2% PEG‐6000 (Sigma Aldrich), 2% Tween‐20 (Sigma Aldrich; Cat. No. P9416), 3% DMSO (New England BioLabs; 12611P), approximately one million molecules of H1‐DBC‐H2 (droplet barcode [DBC]), 0.05 μM of the connector primers H3’‐H2’ and H3 and 0.2 μM of H1 and Bio‐H4’ primers (see Table S2 for sequences). All primers and oligonucleotides for emPCR were purchased from Integrated DNA Technologies (IDT). For each sample, two emulsion reactions were prepared. The protocol for generating water‐in‐oil emulsion droplets is described in detail by Redin et al. (2019 (link)). The emPCRs were performed in a Mastercycler Pro S (Eppendorf) machine, initiated by denaturation at 95°C for 5 min (ramp speed 100%) and then the following cycling profile: 30 cycles of 95°C for 30 s (ramp speed 50%), 60°C for 60 s (ramp speed 20%) and 72°C for 60 s (ramp speed 20%) followed by seven cycles of 95°C for 30 s (ramp speed 50%), 45°C for 60 s (ramp speed 20%) and 72°C for 60 s (ramp speed 20%). The emPCR program ended with 600 s at 72°C (ramp speed 20%) and then hold at 4°C (ramp speed 100%).

The emulsion breakage and library preparation steps were done as previously described by Redin et al. (2019 (link)), except for modifications in the PEG‐based purification. Briefly, following aqueous phase recovery and PCR product purification by 20% PEG (Sigma Aldrich), biotinylated amplicons were enriched using MyOne Streptavidin Dynabeads (Thermo Fisher Scientific). After NaOH treatment, an indexing reaction was performed using a master mix containing Phusion Hot Start Flex (New England BioLabs; Cat. No. M0536L) and 0.2 μM of i5‐H1 and i7‐H4’ indexing primers (see Table S2 for sequences) using the following program: two cycles of 2 min at 95°C, 1 min at 60°C (with 20% ramp speed) and 10 min at 72°C (with 3% ramp speed). The final product was then purified three times using 16% PEG and the quality was examined by Bioanalyzer (Agilent). The libraries were quantified by Qubit fluorometer (Invitrogen) and sequenced using Reagent Kit v3 (150‐cycle) for the MiSeq instrument (Illumina).