ena-EXPERIMENT-Dbitton-16-06-2018-16:49:58:087-12
Illumina MiSeq paired end sequencing
Hermes insertion libraries were constructed as described (Park et al. 2009) using the pHL2577 and pHL2578 plasmids, except that the transposition frequency was calculated by dividing the number of colonies on YES 5-FOA+G418 plates by the number of colonies on YES plates. All experiments were performed in an S. pombe strain with the genotype ura4�D18 leu1�32 h�. Typically, <0.2% of cells in libraries contained genomic Hermes insertions, so we expect that most insertion mutants contain a single insertion.
HERMES_HL_B_2
Genomic DNA was extracted from insertion libraries using phenol/chloroform extraction. All DNA extracted from a library was processed. DNA was sheared to an average size of 200 bp using a Covaris S2 ultrasonicator (Covaris, Woburn, Massachusetts). Sheared DNA was end repaired using the NEBNext� End Repair Module (NEB, Hitchin, UK). Linker1-Random10mer and Linker2 were ligated using the NEBNext� Quick Ligation Module (NEB, Hitchin, UK). In Linker1-Random10mer, the random 10 nt sequence acted as a UMI to distinguish unique chromosomal insertions from PCR amplifications. DNA was then digested with KpnI-HF (NEB, Hitchin, UK) to exclude residual Hermes pHL2577 donor plasmid from PCR amplification (as the plasmid contains a unique KpnI site). NEBNext� modules were used according to manufacturer�s instructions. To enrich for fragments containing the Hermes transposon, DNA was amplified with BIOTAQ� DNA polymerase (Bioline, Essex, UK) using a primer that complimentary to the Hermes transposon (1-Transposon-4NNNN), and to the linker (Linker1-Amp). Ultimately, a second PCR attached the multiplex oligonucleotides for Illumina MiSeq sequencing; the MS-102-2022 MiSeq reagent kit v2 (300 cycles) (Illumina, Cambridge, UK) was used to sequence the libraries. To increase the complexity of the libraries, for each library, ligation and PCR reactions were performed in multiple reactions (in 96-well plates), using a maximum of 1 �g of DNA per well and then re-pooled before sequencing. Detailed protocols are available in the Figshare project Hermes Transposon Mutagenesis of the Fission Yeast Genome.
Dbitton
ena-EXPERIMENT-Dbitton-16-06-2018-16:49:58:087-12