GSM5185713: D2_kpa1-KO_G200; Schizosaccharomyces pombe; OTHER
20 mL of a saturated culture were pelleted (centrifugation 5 minutes at 1600 g) and washed in 1 mL of 10 mM of EDTA pH 8.9. Then, 250 μL of EDTA/ß-mercapto-ethanol/zymolase (EDTA 10 mM pH 8.9, 1% ß-mercapto-ethanol, 2 mg/mL zymolase) were added to the cell pellet and incubated at 37°C for 1 hour with shaking 400 rpm (ThermomixerC – Eppendorf). Equal amount of glass beads acid-washed (Sigma G8772) and cell pellet were added prior to 4 cycles of 15 seconds at 4 m/s on the FastPrep-24 from mpbioTM to help to break cell wall. Following mechanistic cell break, a 10 minutes heat shock at 65°C was performed. gDNA was then extracted and purified according to manufacturer guidelines for the DNeasy Plant Mini Kit from QIAGEN®. Total DNA were used to generate barcoded, Illumina compatible, libraries. Basically, adapters are ligated to DNA followed by an amplification and clean up. The size of the libraries was checked using the Agilent 2100 Bioanalyzer High Sensitivity DNA chip and their concentration was determined using the Qubit® fluorometer (ThermoFisher Scientific). Libraries were applied to an Illumina flow cell for cluster generation (SR Cluster Kit cBot) and sequence-by-synthesis paired reads of 150 base length using the SBS Kit (Illumina) were generated on the HiSeq 2500 following the standard sequencing protocol. Reads were further processed using the bcl2fastq package (Illumina) to split reads according to adapter indexes and produce fastq files. DNA-Seq was performed at Novogene (21F, Tower B, Jinma Building, No.38 Xueqing Road, Haidian District, Beijing, China).
GEO Accession
GSM5185713