Rtl plus buffer

CTCs were released from the Parsortix cassette into ultra-low attachments culture plates. Cells were picked using micromanipulation (CellCelector, ALS) and transferred to RTL Plus buffer (Qiagen) within individual microcentrifuge tubes. Next, genomic DNA was amplified using the MDA method (GenomiPhi V3, GE Healthcare) and subject to Illumina library preparation with SureSelect XT Human All Exon V6 + Cosmic kit (Agilent Technologies). Sequencing was performed on HiSeq 2500 platform (Illumina) with 101 bp paired-end mode. For primary tumour sequencing, three 10 μm-thick paraffin sections from a primary tumour biospy were digested in DNA digestion buffer (50 mM Tris-HCl pH 8.5, 1 mM EDTA pH 8.0 and 0.5% Tween with addition of Proteinase K) in an eppendorf tube at 56 °C for 1 h, followed by 1 h incubation at 90 °C and 5 min at 95 °C. Samples were then kept on ice until RNase A treatment was performed (30 minutes at 37 °C). Next, DNA was precipitated using 7.5 M ammonium acetate and 100% isopropanol. After washing with 70% ethanol, the DNA pellet was air dried and then resuspended in standard Tris-containing buffer. Sequencing was performed on HiSeq 2500 platform (Illumina).

Bovine (Bos taurus) ovaries were collected at a local abattoir and returned to the laboratory in cold PBS. Fully-grown oocytes were removed first by aspirating 3–8 mm surface visible follicles. Subsequently, the ovaries were butterflied open, submerged in cold Dissection medium (TCM199 supplemented with 0.4% BSA fraction V, 0.164 mM penicillin, 0.048 mM streptomycin, 1,790 units/L heparin, and 5 μM cilostamide [26 ]), and growing oocytes from late secondary to early antral follicles were liberated by slicing the ovarian cortex. Subsequently, the medium containing the oocytes was filtered through a descending series of mesh filters, including 260 μm mesh, 100 and 40 μm PluriStrainer® filters. Recovered oocytes were washed in cold Dissection medium, denuded by pipetting, and allocated singly to drops of cold PBS, where their diameter was measured under a Nikon Eclipse TE2000-S microscope and NIS Elements BR 5.02.00 64-bit software. Measured oocytes were snap-frozen in 4 μL PBS + 4 μL RTL-Plus buffer (Qiagen) in 0.2 mL PCR microtubes.