Adapter Preparation

TruSeq v3-style adapters

If you are using custom TruSeq-style sequencing adapters (i.e., not the standard Illumina TruSeq adapters and something similar to those adapters in [Fair2012]) and you have ordered both indexed oligos and a universal oligo from IDT, you need make indexed adapters from the indexed oligo + universal oligo. This means combining one indexed oligo with the universal oligo, and annealing those to make double- stranded adapters. You need to do this for all adapters you will use, and it is best/easiest to do this in a PCR plate.

  1. Assemble the following components to make Oligo Buffer:

    1X TE (10 mM Tris pH 8.0, 1 mM EDTA) 9.9 mL
    5 M NaCl 0.1 mL
    Total 10.0 mL

  2. Using the Oligo Buffer from above, assemble the following components to create the adapters:

    Oligo Buffer 50.0 µL
    100 uM TruSeq Universal Adapter 25.0 µL
    100 uM TruSeq Indexed Adapter 25.0 µL
    Total 100.0 µL

  3. Anneal:

    • 95 C for 1 minute
    • -0.1 C/sec for each of 800 seconds (~13 minutes)
    • 14 C hold

  4. Store adapters at -20 C.