title: Illumina HiSeq 2000 sequencing; Effect of Hamamelitannin, vancomycin and combination treatment of Hamamelitannin and vancomycin on gene expression in S. aureus Mu50 biofilms
design: Effect of Hamamelitannin, vancomycin and combination treatment of Hamamelitannin and vancomycin on gene expression in S. aureus Mu50 biofilms
library_construction_protocol: Staphylococcus aureus Mu50 (MRSA Mu50) biofilms were formed in microtiter plates as previously described (Brackman et al, 2011). Biofilms received no treatment (samples 1A, 1B and 1C), a treatment with HAM (samples 2A, 2B and 2C) or VAN (samples 3A, 3B and 3C) alone or a combination of VAN and HAM (samples 4A, 4B and 4C). 'RNA was extracted immediately following harvesting of the cells, using the Ambion RiboPure Bacteria Kit (Ambion, Austin, TX) according to the manufacturerâs instructions and included a DNase I treatment for 1 h at 37Â°C.Concentration and quality of the total extracted RNA was checked by using the ''Quant-it ribogreen RNA assay'' (Life Technologies, Grand Island, NY, USA) and the RNA 6000 pico chip (Agilent Technologies, Santa Clara, CA, USA), respectively' 'approximately 30 ng of rRNA depleted RNA was used to create barcoded strand specific libraries with the Truseq stranded library preparation kit (Illumina, San Diego, USA).Library preparation was performed using the '' NEBNext Ultra Directional RNA libr prep for Illumina'' (NEB, Ipswich, MA, USA) according to manufacturer''s protocol.Libraries were quantified by qPCR, according to Illumina''s protocol ''Sequencing Library qPCR Quantification protocol guide'', version February 2011. A DNA 1000 chip (Agilent Technologies, Santa Clara, CA, US) was used to control the library''s size distribution and quality. Sequencing was performed on a high throughput Illumina NextSeq 500 flow cell generating 75 bp single reads.'
attributes.tag: Experimental Factor: none
attributes.value: compound