Herculase 2 polymerase

smMIP‐multiplexed amplification was based on Hiatt et al. (2013) using a SensoQuest thermocycler (SensoQuest GmbH), with minor modifications to the protocol. Herculase II Polymerase (Agilent) was used during extension and amplification steps for increased fidelity of microsatellite replication (Fazekas et al., 2010). For amplification, the thermocycler program used 98°C for 2 min, 30 cycles of 98°C for 15 s, 60°C for 30 s and 72°C for 30 s, followed by 72°C for 2 min. Hundred nanograms of sample DNA was used as a template unless stated otherwise: The input quantity of CRC sample DNA varied depending on quantity available (Table S1). smMIP reaction products (240–270 bp) were analyzed using 3% agarose gel electrophoresis at 80 mV for 60 min, or a QIAxcel (Qiagen).

Genomic DNA (gDNA) was prepared from overnight cultures of single reduced-capsule mutants using a DNeasy blood and tissue kit (Qiagen). Random-prime PCR to identify the transposon insertion site in each gDNA template was performed as previously described (75 (link)) using primers FS57-59 and FS109 and Herculase II polymerase (Agilent). Amplicons were sequenced using primer FS107.

Capture-C was performed as described previously^{80 (link),81 (link)} for three biological replicates per experimental condition. Briefly, 10 × 10⁶ cells per biological replicate were crosslinked, followed by cell lysis. 3 C libraries were generated by DpnII digestion and subsequent proximity ligation. After decrosslinking and DNA extraction, the resulting 3 C libraries were sonicated to a fragment size of ~200 bp and indexed with Illumina sequencing adapters, using Herculase II polymerase (Agilent, 600677) for library amplification. To boost library complexity, indexing was performed in two parallel reactions for each sample. Biotinylated oligonucleotides (70 nt) were designed using a python-based oligo tool^{82 (link)} (https://oligo.readthedocs.io/en/latest/) and used for enrichment of the libraries in two consecutive rounds of hybridization, biotin-streptavidin bead pulldown (Invitrogen, 65306), bead washes and PCR amplification (KAPA HyperCapture Reagent Kit, Roche, 09075828001). The final libraries were assessed on a fragment analyzer and sequenced using the NextSeq550 Illumina platform (75-bp paired-end reads). Data analysis was performed using the CapCruncher pipeline^{80 (link)} (https://github.com/sims-lab/CapCruncher).