GSM2418825: Gen0 - trial 1 Sample 2; Yarrowia lipolytica; Tn-Seq
DNA was isolated by standard phenol:chloroform After extraction, gDNA was treated with DNase-free RNaseA (100 µg/mL) (Sigma- Aldrich) at 37° C for 4 hrs, followed by Pronase (250 µg/mL) (Sigma-Aldrich) at 37°C for 2 hrs. After both RNase and Pronase treatment, another phenol:chloroform extraction was performed. Approximately 30 µg of this treated DNA was sheared to less than 1 kb in a 100 µl volume using the Biorupter Standard (Diagenode). DNA size was confirmed by gel electrophoresis. Sheared DNA was concentrated using the Clean and Concentrator with 5X ChIP DNA Binding Buffer (Zymo Research). DNA ends were made blunt using the Fast DNA End Repair kit (Thermo Scientific); 3’ adenylated using the Klenow Exo-enzyme with 0.2 mM dATP (Fisher Scientific). Custom sequencing adapters (0.5 µM) with Illumina multiplex barcodes (Additional file 1: Table S1) were ligated to DNAs in each sample using T4 ligase (Thermo Scientific) by incubating at 22°C for 12 hrs followed by 65°C for 10 min. Between enzyme reactions, samples were purified using the Agencourt AMPure XP purification beads (Beckman Coulter, Inc.). Hermes TIR –gDNA borders were enriched in PCR using the high-fidelity KOD polymerase (EMD Millipore) as follows: 95°C 5 min, 5 cycles of 95°C 30 s, 65°C 30 s, and 70°C 30 s, followed by 20 cycles of 95°C 30 s, 61°C 30 s, and 70°C 30 s, and a final 70°C incubation for 2 min. Forward primer KP5113 anneals 26 bp from the downstream 3’-end of the TIR, and reverse primer KP5112 anneals to the adapter upstream of the Illumina multiplex barcode. After sizing/purification of PCR products samples were submitted to the UCI Genomics High-throughput Facility for Bioanalyzer 2100 (Agilent) analysis, KAPA quantification and single-end 100 cycle sequencing on the Illumina HiSeq 2500.
GEO Accession
GSM2418825