Bstyi

RT-PCR detection of PRKCA^D463H mutant cDNA: cDNA were retrotranscribed from tumor RNA samples (1 µg) with Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Fisher Scientific K1641) according to manufacturer instructions, then amplified with PCR primers VG-229 and VG-623 by using Fast Start PCR Master (Roche 04710452001). PCR reactions were split and half was digested with BstYI (New England Biolabs), when the other half was incubated in the digestion mix without BstYI, and left over night at 60 °C. Fragments were analyzed on 2% agarose gel or by using LabChip GX bioanalyzer (Caliper LifeSciences, Villepinte, France).
PRKCA RT-qPCR was performed using LightCycler^® 480 Probes Master mix and Universal Probe Library^TM probes specific to each genes. Primers and probe number are indicated in Supplementary Data 11. The reference primers amplified PPIA (Peptidylprolyl isomerase A = Cyclophilin A). Real-time qPCR reactions were performed according to the manufacturer’s instructions. The 2^−ΔCT method was used to determine the relative expression, where ΔCT = CT_{target gene}–CT_PPIA.
PRKCa^WT cDNA synthesis for plasmid construction (see below): RNA was extracted from a glioblastoma spheroid culture, non-mutated on PRKCA. First Strand cDNA Synthesis Kit (Fisher Scientific, Illkirch, France) was then used to generate cDNA.

Whole-genome amplified DNA samples were digested with AseI, BspHI, BstYI, HindIII, NcoI and PstI (New England Biolabs). Alternatively, the whole-genome amplified DNA was end-repaired by 5′ phosphorylated and 3′ dA tailing using the NEBNext Ultra II End Repair/dA-Tailing Module (New England Biolabs, cat no. E7546S). A pair of vectorette oligonucleotides (synthesized by IDT) corresponding to each restriction enzyme or T tail were annealed to form vectorette adaptors with the sticky end created. See sequences reported in [47 (link)]. Then the digested or repaired amplified genomic DNA were ligated with the vectorette adaptors using T4 DNA ligase (New England Biolabs, M0202S) at 4°C overnight. After ligation, PCR was performed with the L1 primer (5′- AGA TAT ACC TAA TGC TAG ATG ACA CA -3′) and the Vectorette Primer (5′- CTC TCC CTT CTC GGA TCT TAA -3′) using ExTaq (Takara, cat no. RR006A) with a touchdown programme (95°C 5 min; 95°C 1 min, 72°C 1 min, 72°C 5 min, 5 cycles; 95°C 1 min, 68°C 1 min, 72°C 5 min, 5 cycles; 95°C 45 s, 64°C 1 min, 72°C 5 min, 15 cycles; 95°C 45 s, 60°C 1 min, 72°C 5 min, 15 cycles; 72°C 15 min; 4°C hold).

Chromosome conformation capture (3C) analysis was performed on control and 3′ WRE-Mut cell populations as previously described with minor modifications [38 (link)]. A bacterial artificial chromosome harboring the MYC genomic locus served as a control for the 3C-qPCR analysis. Formaldehyde cross-linked chromatin was digested with 40 μL (400 units) of BstYI (New England Biolabs) overnight at 37 °C. Primer sequences used to detect the MYC 5′3′ chromatin loop, and those to amplify the control region, are available in Table S1. 3C products were amplified for 32 cycles in triplicate reactions using DreamTaq polymerase (ThermoFisher, K1081). Products were resolved on a 1.5% agarose gel, imaged, and band intensities were quantified using ImageJ software. Each MYC 3C product was first normalized to the average value obtained from the three BAC 3C products, then to the average value of the three internal control bands.