Seramag

Primers were designed to amplify a 150–250 bp region surrounding the cut site. Genomic DNA was amplified in two rounds using NEBNext Ultra II Q5 HiFi polymerase (New England Biolabs). In the first round, Illumina adapter sequences were included at the ends of the primers amplifying the genomic DNA in 20 reaction cycles. In the second round, 1 µL from the first PCR was used as input and i7 / i5 Illumina indexes were added in 10 reaction cycles. Common amplicons were then pooled and purified using 0.8x SPRI beads (SeraMag; Cytiva). Concentrations were measured on a Qubit and further analyzed for amplicon length and sample purity on a TapeStation high-sensitivity DNA flow cell (Agilent). Pools were then normalized and combined. The combined samples were sequenced either with a MiSeq 2 × 100 paired-end or a NextSeq2000 2 × 150 paired-end (Illumina) by the Functional Genomics Centre Zurich (FGCZ) in combination with the Genome Engineering and Measurement Lab (GEML) at ETH Zurich. The target average read count per amplicon was 100–200k reads.

Primers were designed to amplify a ~6 kb region surrounding the expected cut site. 600 ng of genomic DNA (corresponding to ~1 × 10⁵ cells) was amplified using Q5 (Q5 High-Fidelity DNA Polymerase, New England Biolabs) with an annealing temperature of 60 °C, extension time of 3 min 30 s, and a cycle number of 30. Common amplicons were then pooled and purified using 0.5x SPRI beads (Sera-Mag; Cytiva). Purified amplicons were then given to Functional Genomics Centre Zurich (FGCZ) for indexing. Indexed amplicons were analyzed on a PacBio Sequel IIe with a SMRT Cell 8 M, 30 h movie.

Co-IP lysates were prepared as above using 40 μL α-FLAG-conjugated magnetic beads. Following Co-IP, samples were resuspended in Co-IP buffer with reduced Triton X-100 (0.1%). To prepare samples for mass spectrometry, FLAG-tagged affinity purifications were eluted, reduced, and alkylated using 5% (w/v) sodium dodecyl-sulfate, 10 mM tris(2-carboxyethylphosphine), 40 mM 2-chloroacetamide, 50 mM Tris-HCl, pH 8.5, boiled for 10 min, and incubated shaking at 1000 rpm at 37°C for 30 min. Affinity-purified proteins were digested using the SP3 method with Sera-Mag™ carboxylate-functionalized SpeedBeads (Cytiva).^{54 (link)} Cleaned-up peptides were then dried in a SpeedVac vacuum concentrator and stored at −20°C until analysis.
Tryptic peptides were suspended in 3% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid and directly injected onto a reversed-phase C18 1.7 μm, 130 Å, 75 mm × 250 mm M-class column (Waters), using an Ultimate 3000 nanoUPLC (ThermoFisher). Peptides were eluted with an acetonitrile gradient and detected using a Q-Exactive HF-X mass spectrometer (ThermoFisher). Raw files were searched against the Uniprot Human database UP000005640 using MaxQuant v.1.6.14.0. All peptide and protein identifications were thresholded at a 1% false discovery rate. Statistical analysis was performed on log2-transformed iBAQ intensities using limma.