Bar-seq library time point 0 replicate A_1
In the first PCR step UPTAGs from a single sample were amplified with the primers Illumina UPTAG Index # (5'-ACG CTC TTC CGA TCT NNNNN GTC CAC GAG GTC TCT-3') and Illumina UPkanMX (CAA GCA GAA GAC GGC ATA CGA GAT GTC GAC CTG CAG CGT ACG-3') and DNTAGs from the same sample were amplified with the primers Illumina DNTAG Index # (5'-ACG CTC TTC CGA TCT NNNNN GTG TCG GTC TCG TAG-3') and IlluminaDNkanMX (5'-CAA GCA GAA GAC GGC ATA CGA GAT ACG AGC TCG AAT TCA TCG-3') in separate PCR reactions. Illumina UPTAG and Illumina DNTAG primers contain a 5 base pair sequence (denoted NNNNN in the primer sequence) that uniquely identifies the sample. We designed 120 unique indices that differ by at least two nucleotides. A complete list of primer sequences is provided in Table S1. We normalized genomic DNA concentrations to 10ng/μL and using 100ng of template amplified barcodes using the following PCR program: 2 minutes at 98 ◦C followed by 20 cycles of 10 seconds at 98 ◦C, 10 seconds at 50◦C and 10 seconds at 72◦C and a final extension step of 2 minutes at 72◦C. PCR products were confirmed on 2% agarose gels and purified using Qiagen PCR cleanup kits. We quantified purified PCR products using a Qubit fluorimeter and combined 60ng from each of the 20 different UPTAG libraries and, in a separate tube, 60ng from each of the 20 different DNTAG libraries. The multiplexed UPTAG libraries were then amplified using the primers P5 (5'-A ATG ATA CGG CGA CCA CCG AGA TCT ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TCT-3') and Illumina UPkanMX and the combined DNTAG libraries were amplified using the P5 and IlluminaDNkanMX primers using the identical PCR program as the first step with 20ng of template. The 140 base pair UPTAG and DNTAG libraries were purified using Qiagen PCR cleanup columns, quantified using a Qubit fluorimeter, combined in equimolar amounts and adjusted to a final concentration of 10nM (i.e. 0.924 ng/μL). The library was sequenced on a single lane of an Illumina HiSeq 2000 using standard methods including the use of the standard Illumina sequencing primer (5'-ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TCT-3').