Ion torrent proton platform

Gene expression analyses were performed as previously described^{38 (link)}. In brief, RNA was isolated from bulk tumor fragments, and sequenced on an Ion Torrent Proton platform (Thermo Fisher) using a mouse-specific Ion AmpliSeq Custom Panel designed to target 3,827 cancer-related genes. Counts were generated using Torrent Suite and AmpliSeqRNA analysis plugin (Thermo Fisher). For GSEA, we used the MSigDB Hallmarks v6.1 dataset with weighted enrichment statistic and signal-to-noise metric for ranking genes.

Deep APC sequencing was performed using a previously described custom APC panel [27 (link)]. The complete sequencing panel consisted of 115 amplicons (11,216 bp), covering 99.3% of the coding regions of APC. Libraries were prepared with Ion Ampliseq™ 2.0 Kit (Thermo Fisher Scientific, Bleiswijk, The Netherlands) according to the manufacturer’s instructions and were sequenced on the Ion Torrent Proton Platform (Thermo Fisher Scientific, Bleiswijk, The Netherlands). Sequence data were analyzed as described previously [27 (link)]. Variants were annotated to the GenBank reference sequence NM_000038.4. The Integrative Genomics Viewer (IGV) (https://www.broadinstitute.org/igv/) was used to visualize the read alignment and the presence of variants against the reference genome.

Psyttalia humilis and P. lounsburyi were sequenced using the Ion™ Torrent Proton™ platform (ThermoFisher Scientific, Waltham, MA, USA) available at the Central Analytical Facilities of Stellenbosch University, South Africa. Sequence libraries were prepared using the NEXTflex™ DNA Sequencing Kit for Ion Platforms (PerkinElmer, Waltham, MA, USA) according to the BI00 Scientific v15.12 protocol. Libraries were diluted to a target concentration of 60 pM. The diluted, barcoded libraries were combined in equimolar amounts for template preparation using the Ion PI™ Hi-Q™ Chef Kit (Thermo Fisher Scientific, Waltham, MA, USA). Twenty five microliters of diluted, pooled library was loaded onto the Ion Chef liquid handler (Thermo Fisher Scientific) for template preparation and enrichment using Ion PI™ Hi-Q™ Chef reagents, solutions and supplies according to the protocol, MAN0010967 REVB.0. Enriched ion sphere particles were loaded onto an Ion PI™ v3 chip. Massively parallel sequencing was performed on the Ion Torrent Proton system using sequencing solutions, reagents and supplies according to the protocol MAN0010967 REV B.0. Flow space calibration and basecaller analysis were performed using standard analysis parameters in the Torrent Suite version 5.10.0 software.

FACS-sorted MuSCs were cross-linked with 1% formaldehyde/PBS for 10 min, before quenching with 0.125 M glycine. Cross-linked cells were washed with ice-cold PBS for three times and spun down. Chromatin preparation and protein-DNA complex immunoprecipitation was carried out according to established protocols^{68 (link)}. ChIPed DNA was purified using mini-elute PCR kits (Qiagen). DNA concentration was measured using Qubit® Fluorometric Quantitation (Life Science). ChIP-seq libraries were generated using the Ion ChIP-Seq Library Preparation Kit (Thermo Fisher) following standard protocols. Libraries were analyzed with the Bioanalyzer 2100 and sequenced using the IonTorrent Proton platform with Ion PI Sequencing 200 Kit v2 (Thermo Fisher). Resulting raw reads were assessed for quality, adapter content and duplication rates with FastQC. Data processing of sequencing reads in FASTQ format were performed as described above for mES cells. All downstream analyses were carried out with R/BioConductor (http://www.bioconductor.org) using the packages GenomicRanges and GenomicFeatures. In order to compare H3K56ac to other histone modifications public data sets for H3K4me3 and H3K27ac in MuSCs^{69 (link)} were downloaded from GEO (time-point T3 from GSE103163: GSM2756408, GSM2756400 and GSM2756402).