title: Tn-seq of S. aureus: CSD sort, third enrichment
design: Following isolation of cell pellets by centrifugation, cells were lysed with lysostaphin, then treated with RNAse A (ProMega) and Proteinase K (NEB).  Genomic DNA was sonicated (Q800R2 QSONICA) to 200600 bp fragments, then PolyC-tails were added by incubating fragments with a 20:1 mixture of dCTP (Thermo Scientific) to ddCTP (Affymetrix) and TdT enzyme in TdT buffer (ProMega). This DNA was then subject to an initial round of PCR amplification with primers oTB535 and oTB536 with the Easy A cloning kit (Agilent), digested with NotI (NEB) to remove contaminating genomic DNA, and subjected to a second PCR amplification with primer oTB537 (an Illumina sequencing primer) and a barcoding primer (unique for each sample to allow for demultiplexing of reads) with the Easy A cloning kit (Agilent). A 200400 bp product was gel-purified and sequenced on an Illumina NextSeq Platform (in lab) or an Illumina HiSeq 2500 platform (TUCF Genomics Facility, Tufts University). Following sequencing, reads were mapped to reference genome NC_007795