ICE junction sequencing of WildR gut microbiome: WildR-T6-fitness, WildR, rep2, day 7 feces, mouse 2, 3 prime end of the ICE
Genomic DNA was extracted from fecal samples using mechanical disruption followed by phenol and column clean up. ICE junction libraries ("ICE-seq") were generated following a previously described method for Tn-seq (DOI: 10.1007/978-1-4939-9118-1_12) with the following modifications. DNA was sheared to ~500 bp average size and C-tailed. Sequences outside of the ICE at either the 5' or 3' ends of the ICE ("ICE junction") were specifically amplified using primers annealing within the ICE at the 3' end (primer sequence 5' - GGAAATGCACTCCCCAAGACAAGTAAATGA) and a C-tailed specific primer. After DNA purification, a second round of PCR was performed to amplify the ICE junctions and add adapter and index sequences for sequencing. The primer sequence specific for the ICE 3' end was: 5' - AATGATACGGCGACCACCGAGATCTACACCATTTTATAATATACTGATTATAAGTGCATTGCGGTGATATTTTTATGACAGTTTTG. Samples were pooled and multiplexed samples were sequenced as 75-bp single-end reads on an Illumina MiniSeq with 30% PhiX DNA spiked in using a custom mix containing three primers for sequencing the 5' (5' - TCCTGTATCTTGTTTTCATTTTACACACATCTATAAG), 3' (5' - AAGTGCATTGCGGTGATATTTTTATGACAGTTTTG), and PhiX sequences. Sequencing was performed twice (technical replicates) to generate enough read coverage of the ICE junctions.