GSM5822473: Output 2C; Saccharomyces cerevisiae; OTHER
The input and output pellets (3 biological x 5 technical replicas, 60 tubes in total) were thawed and resuspended in 2ml extraction buffer (2% Triton-X, 1% SDS, 100mM NaCl, 10mM Tris pH 8, 1mM EDTA pH 8), and underwent two cycles of freezing and thawing in an ethanol-dry ice bath (10 min) and at 62C (10min).  Samples were then vortexed together with 1.5ml of phenol:chloroform:isoamyl 25:24:1 and 1.5g of glass beads (sigma). The aqueous phase was recovered by centrifugation and mixed again with 1.5ml phenol:chloroform:isoamyl 25:24:1. DNA precipitation was performed by adding 1:10V of 3M NaOAc and 2.2V of 100% cold ethanol to the aqueous phase and incubating the samples at -20C for 1h. After a centrifugation step, pellets were dried overnight at room temperature. Pellets were resuspended in 1ml resuspension buffer (10mM Tris pH 8, 1mM EDTA pH 8) and treated with 7.5ul RNase A (Thermo Scientific) for 30min at 37C. The DNA was finally purified using 75ul of silica beads (QIAEX II Gel Extraction Kit, Qiagen), washed and eluted in 375ul elution buffer. PCR amplification in two steps using Q5 High-Fidelity DNA Polymerase (NEB). In step 1, 50 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. Other (DNA-seq of PCR amplicon)