title: GSM2674965: Yersinia pestis transposon mutant library cultured at 37ºC (replicate 3); Yersinia pestis; OTHER
library_construction_protocol: genomic DNA extracted using the Gentra Puregene kit (Qiagen) The gDNA was fragmented to <500 base pairs (bp) using 2 x 15 minute cycles at 4ºC in a BioRuptor sonicator (medium intensity, 30s on/90s off).  A NEBNext DNA library preparation for Illumina kit (NEB) was used according to the manufacturer's instructions, to end repair, A-tail and ligate adapters to the fragments.  Parallel polymerase chain reaction (PCR) samples were set up with 10 μl JumpStart 10x buffer, 6 μl MgCl2, 2 μl 10mM nucleoside triphosphates (dNTPs), 0.6 μl 100 μM PE_PCR_V3.3 primer, 0.6 μl 100 μM Yp_EzTn_PCR primer, 1 μl JumpStart Taq DNA polymerase and 28.8 μl nuclease-free water per reaction.  Primer sequences are listed in Additional file 2, Table S2.  The reactions were amplified at 94oC for 2 minutes, (94oC for 30 seconds, 60oC for 20 seconds, 72oC for 30 seconds) for 22 cycles, 72oC for 10 minutes, then held at 12oC.  PCR products were pooled and ethanol precipitated before being size selected on a 2% (w/v) agarose tris-borate-EDTA (TBE) gel.  Agarose blocks corresponding to 350-500 bp were excised, and the DNA extracted using a Qiagen MinElute Gel Extraction kit as per the manufacturer's instructions The DNA was quantified by qPCR and on an Agilent BioAnalyzer before being submitted for sequencing as 100 bp single end reads on an Illumina HiSeq 2500 standard model.
attributes.tag: GEO Accession
attributes.value: GSM2674965