8.1 Fragmentation, End Repair & A-tailing

  1. Prepare a thermal cycler with the following incubation protocol.

    Lid Temperature

    Reaction Volume

    Run Time
    hh:mm:ss

    65°C

    50 µl

    ~35 min

    Step

    Temperature

    Time

    1. Pre-cool block

    Pre-cool block prior to preparing the Fragmentation Mix

    4°C

    Hold

    2. Fragmentation

    32°C

    00:05:00

    3. End Repair & A-tailing

    65°C

    00:30:00

    4. Hold

    4°C

    Hold

  1. Vortex Fragmentation Buffer. Verify there is no precipitate.

  2. Prepare Fragmentation Mix on ice. Pipette mix and centrifuge briefly.

    2

     

    Fixed percentage added to total to help with pipetting

    Fragmentation Mix
    Add reagents in the order listed

    PN

    Volume for Master Mix (μl)

    Scaled Volume (μl)
    Buffer EB - 25

     

    Fragmentation Buffer

    2000091/2001316

    5

     

    Fragmentation Enzyme

    		 2000090/2000104/2001315 10

     

    Total

    		 

    40

     

  3. Transfer ONLY 10 μl purified cDNA sample from cDNA Cleanup (step 7.2n) to a tube strip.

    Note that only 10 μl (25%) cDNA sample is sufficient for generating 3ʹ Gene Expression library. The remaining 30 μl (75%) cDNA sample can be stored at 4°C for up to 72 h or at −20°C for up to 4 weeks for generating additional Gene Expression libraries.

  4. Add 40 μl Fragmentation Mix to each sample.

  5. Pipette mix 15x (pipette set to 35 µl) on ice. Centrifuge briefly.

  6. Transfer into the pre-cooled thermal cycler (4°C) and press “SKIP” to initiate the protocol.