GSM3666032: Output 5B [bTDP1-OU-5B]; Saccharomyces cerevisiae; OTHER
Cell pellets  were resupended in 1.5 mL extraction buffer (2% Triton-X, 1% SDS, 100mM NaCl, 10mM Tris-HCl pH8, 1mM EDTA pH8), frozen by dry ice-ethanol bath and incubated at 62ºC water bath twice. Subsequently, 1.5mL of Phenol/Chloro/Isoamyl 25:24:1 (equilibrated in 10mM Tris-HCl, 1mM EDTA, pH8) was added, together with 1.5 g of glass beads and the samples were vortexed for 10 min. Samples were centrifuged at RT for 30 min at 4,000 rpm to phase separate and the aqueous phase was transferred into new tubes. The same step was repeated twice. 1:10V of NaOAc 3M and 2.2V of cold ethanol 100% were added to the aqueous phase. The mix was incubated at -20ºC for 30 min and after that, centrifuged for 30 min at full speed at 4ºC to precipitate the DNA. The ethanol was removed and the DNA pellet allowed to dry overnight at RT. DNA pellets were resuspended in 900 µL TE 1X and treated with RNAseA (10 mg/mL, Thermo Scientific) for 30 min at 37ºC. To desalt and concentrate the DNA solution, QIAEX II Gel Extraction Kit was used (75 µL of QIagen beads). Finally the sample was washed 3 times with PE and eluted twice in 375 10 mM Tris·Cl, pH 8.5 PCR amplification in two steps using Q5 High-Fidelity DNA Polymerase (NEB). In step 1, 300 million plasmids were amplified for 15 cycles using frame-shifted adaptor primers with a partial homology to standard Ilumina sequencing primers. PCR products from the first step were used as templates in the second PCR step, where indexed Illumina primers were used for a 10 cycles amplification. DNA concentration was then quantified by means of a Quant-iT™ PicoGreen® dsDNA Assay Kit (Promega). All replicates were pooled together in an equimolar ratio. Finally, the pooled sequencing library was run on a 2% agarose gel, purified and sent for 125 bp paired-end Illumina sequencing.
GEO
GSM3666032