Masterpure kit

The HPV testing methodology for the HeNCe Life Study has been previously described [7 (link),16 ]. Cell suspensions were centrifuged at 13,000× g for 15 min at 22 °C. Pellets were resuspended in 300 μL of 20 mmol/L Tris buffer (pH 8.3) and DNA was purified using the MasterPure™ Kit (Epicenter). Extracted DNA was kept frozen until tested with PCR at −70 °C. We used several molecular techniques for testing and genotyping HPV genera. All HPV DNA samples were tested for β-globin, with positive samples considered for further HPV genotyping and negative samples considered inadequate for PCR. A polymerase chain reaction (PCR) assay with PGMY09-PGMY11-primers and linear array was used to test for α-HPV genotypes. This is capable of detecting 36 mucosal genotypes. To detect cutaneous genotypes, β- and γ-HPV, we used multiplex PCR with bead-based Luminex technology. This technique is capable of detecting 43 β-HPV genotypes and 52 γ-HPV genotypes [17 (link)].

Pollen nuclei of wild-type (Col-0) and mutant dme/+, ros1, and dme/+;ros1 plants were purified by FACS using SYBR Green staining as previously described^{49 (link),50 (link)}. Briefly, open flowers were collected into a 50 mL falcon tubes and vortexed in 10 mL of Galbraith buffer (45 mM MgCl₂, 30 mM Sodium Citrate, 20 mM MOPS, 1% Triton-100 pH 7.0) for 3 min, at room temperature. This crude fraction was then filtered through Miracloth (Calbiochem) and centrifuged for 1 min at 2600 × g to concentrate the pollen fraction. The pollen was then transferred to a 1.5 mL Eppendorf tube containing ~100 μL of acid-washed glass beads (425–600 μm, Sigma) and vortexed continuously at maximum speed for 3 min, to break the pollen cell wall. The fraction containing the released nuclei was then filtered through a 10 μm mesh (Celltrics, Sysmex-Partec) to exclude pollen debris and stained with SYBR Green dye (Lonza). FACS was performed using a FACSAria IIU cell sorter (BD Biosciences), using the integrated FACSDiva v6.1 software with 70 µ nozzle at 70 psi. A 488 nm laser was used for SYBR Green excitation, which was detected by a 530/30 nm band-pass filter (Supplementary Fig. 2). Approximately 500,000 nuclei from each genotype and cell type were purified, and genomic DNA was extracted using the Masterpure kit (Epicenter).

Genomic DNA was extracted using a MasterPure kit (Epicentre, USA) from the concentrated bead samples. After defrosting, cells attached to sampled beads were lysed using a buffer containing 10 mM Tris–HCL and 1 mM EDTA and 5 μl of 50 mg ml⁻¹ lysozyme and incubating for 30 min at 37°C, after which the extraction protocol was followed. DNA was precipitated by incubation of the samples with glycogen for 30 min at −20°C prior to centrifugation and washing. DNA from 1/32 of the SPM filters was extracted using the Powersoil® DNA Isolation kit according to the manufacturer's instructions (MoBio, Qiagen, Carlsbad, CA), except with an elution step with 50 μl TE. DNA concentrations were measured fluorometrically with Qubit™ dsDNA HS assay kit (Thermo Fischer Scientific, Waltham, MA), and extracts were stored at −80°C.