Biotin 11 ddatp

Genomic DNA was fragmented by incubating at 37°C for 1 min. in a 20 μl reaction containing 1 × One-Phor-All Plus Buffer (GE Healthcare) and 0.01 units DNase I (GE Healthcare) as previously described (Jackson et al., 2011 (link)), and further modified by Tall et al. (2015 (link)). The fragmented DNA was heat-inactivated at 99°C for 15 min, and was 3′-end labeled by adding 4 μl of 5 × terminal transferase buffer (Promega), 1 μl 1 mM biotin-11-ddATP (PerkinElmer NEL508), and 2 μl (60 units) of terminal transferase enzyme (Promega). The labeling was carried out for 4 h at 37°C followed by heat inactivation at 98°C for 1 min.
Hybridizations were performed according to the Affymetrix GeneChip Expression Analysis Technical Manual for a 49-format array (Affymetrix, 2014 ). Following hybridization, wash and stain procedures were carried out on an Affymetrix FS-450 fluidics station using the mini_prok2v1_450 fluidics script (Affymetrix, 2014 ). Reagents for washing and staining were prepared according to the GeneChip® Expression Analysis Technical Manual (Affymetrix, 2014 ). The following modifications were made to the wash and stain procedure: Streptavidin solution mix (vial 1) was replaced with SAPE solution mix (Life Technologies, Grand Island, NY). Arrays were scanned using Affymetrix GeneChip® Scanner 3000 running on AGCC software.

Thirty types of poly-A tailed DNA barcode molecules were prepared by referring to the sequence of Helicos control oligo. Firstly, we added a poly-A tail to the oligonucleotides (Supplementary Table 1) by incubating 10 µl of mixture consisting of 10 µM oligonucleotide mix, 1× TdT buffer, 250 µM CoCl₂, 240 µM dATP, and 1 U/µl of terminal transferase at 37 °C for 1 h and 70 °C for 10 min followed by a 4-°C hold. Secondly, the sample was added to 10 µl of mixture consisting of 1× TdT buffer, 250 µM CoCl₂, 160 µM biotin-11-ddATP (PerkinElmer), and 2 U/µl terminal transferase (NEB) and incubated at 37 °C for 1 h, and 70 °C for 10 min followed by a 4-°C hold.

The FDA Salmonella custom high-density Affymetrix DNA microarray platform was used, as previously described [34 (link),35 (link)]. An 8 μg aliquot of purified genomic DNA was fragmented by incubation at 37 °C for 10 min in a 50 μL reaction containing 1× One-Phor-All Plus Buffer [Tris, Magnesium and Potassium acetate (Ratios 1:1:5)] and 0.1 units DNase I (GE Healthcare, Pittsburg, PA, USA). Following fragmentation, the DNA was labeled at the 3′-end using 1 mM biotin-11-ddATP (PerkinElmer NEL508, Waltham, MA, USA), 5X terminal transferase buffer (Promega, Madison, WI, USA), and 60 units of terminal transferase enzyme (Promega), as described earlier. The genomic DNA samples were hybridized following the Affymetrix GeneChip Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA, USA, 2014), washed in the Affymetrix FS-450 fluidics station (Affymetrix, Santa Clara, CA, USA), and scanned using software of the Affymetrix GeneChip Command Console (AGCC) Scanner 3000. Reagents used in hybridization, washing, and staining were prepared according to the Affymetrix GeneChip Expression Analysis Technical Manual [36 ]. For microarray data analysis, a probe set intensity for each allele represented on the microarray chip were assessed using the Robust MultiArray Averaging (RMA) function in the Affymetrix package of R-Bioconductor [37 (link)].