GSM8464297: MP1_subset28_pool1_S1; Saccharomyces cerevisiae; OTHER
In this method we extract plasmid DNA from yeast, serving as a template for by a PCR reaction which is the first step of the library preparation protocol. Plasmid DNA was extracted using the MasterPure Yeast DNA Purification Kit (Lucigen Corporation, MPY80200), with some modifications. Briefly, 300 ul of lysis buffer supplemented with RNase A (as recommended) were added to each sample. Following 15 minutes of incubation in 65 C, cells were moved into LoBind microcentrifuge tubes (Eppendorf®, 022431021) containing 0.5mm Zirconium Oxide beads (ZrOB05, Next Advance) and blended using the Bullet Blender 24 (Next Advance) for a single cycle of 3 minutes in level 8. Samples were cooled over ice and lysates were moved to 1.5 ml tubes, vortexed, and supplemented with 300 ul MPC buffer. Samples were vortexed vigorously and centrifuged for 10 minutes (17,000g). Supernatant was moved to 1.5 ml tubes containing 500 ul isopropanol. Samples were vortexed vigorously and DNA was precipitated (10 minutes, 17,000g). Isopropanol was removed, and pellets were washed in 70% EtOH, then air dried and finally resuspended in TE buffer. Following DNA extraction, samples were 1.5 X SPRI (AMPure XP, A63881) cleaned and resuspended in 30 ul elution buffer (10mM Tris-HCl, pH 8) and then diluted 1:20. 1 ul was used as a template for PCR reaction for amplifying noncleaved variants and sample barcoding (28 cycles) using the Phusion® High-Fidelity PCR Master Mix (NEB, M0531L). Samples were verified using gel electrophoresis, and 0.5 reversed-SPRI cleaned. DNA concentrations were measured using Qubit™ Flex Fluorometer (Invitrogen), and samples were normalized to equal amounts, then pooled together. 1 ng was taken as a template for a PCR reaction using KAPA HiFi HotStart ReadyMix (Roche, KK2602) for adding iIlumina indices and machine adapters. Samples were 1X SPRI cleaned, then concentrations and library quality were validated using Qubit and tape station (Agilent).