Instrument: Illumina MiSeq
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: PAIRED
Construction protocol: NFR-DNAs were rescued from either the initial lentiviral plasmid libraries or gDNA of GFP+ selected cells using PCR in a method adapted from bacterial rRNA sequencing7. In this method, Illumina sequencing adaptors are included in the primers, permitting one step amplification and sequencing library preparation. Primers (termed FMS-F/R,) were designed such that they contain recognition sequences complimentary to lentiviral sequence flanking NFR-DNA and Illumina adaptor sequence for paired-end sequencing in addition to a 6base pair Index sequence. Six PCR reactions (10 ul Phusion Polymerase buffer, 1 μl 10 mMdNTP, 2.5 μl 10 μM forward and reverse primer, 1.5 μl DMSO, 0.5 μl (NEB) 50 ng DNA, 31 μl H20; 16 cycles with 550C annealing temperature) per plasmid library were pooled and sequenced. FIREWACh elements were recovered from the genomic DNA of a least 1x106 FACS-sorted GFP+ cells using PCR. In this case 10 PCR reactions were performed using the same conditions as above but 100 ng gDNA and 23 cycles of amplification. The 10 reactions were then pooled. Six PCR reactions (10 ul Phusion Polymerase buffer, 1 μl 10 mMdNTP, 2.5 μl 10 μM forward and reverse primer, 1.5 μl DMSO, 0.5 μl (NEB) 50 ng DNA, 31 μl H20; 16 cycles with 550C annealing temperature) per plasmid library were pooled and sequenced. Quality was assessed determining size distrubution with with bioanalyzer and concentration was confirmed via QPCR Up to six samples were pooled within a single lane on a MiSeq machine. Input library derived NFRs were sequenced together using three of the indices while FIREWACh NFRS, i.e. NFR’s rescued from GFP+ cells, were run with six samples per lane, each sample representing NFRs rescued from an independent biological replicate (i.e. HaeIII_BioRep1 etc). Technical replicates consisting of independent PCR rescued NFR sequencing libraries were sequenced on separate days.