ChIP-Seq libraries

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Sequencing library preparation for ChIP samples
Date:
Name:
Prepare: 2% LRA gel, 5 samples per gel

[править] Sample Preparation

NB! All reaction in LB tubes


  1. Sonication UTR200: 1hour; 100% amplitude; 0.5 cycle. Samples should be in 1xTE

    Sample numbermQ water10xTEDNA
    1
    2
    3
    4
    5
    6
    7
    8



    • Zymo purification -- elute in 10μl EB



  2. End-repair

    ER-mix 9.08x2.2
    mQ6.1
    ER enzyme mix 12x1.7
    DNA10
    total20µlx


    Water bath: 30min -- 20’C
    • Zymo purification -- elute in 10μl EB



  3. A-tailing

    A-tailing buffer 8.33x2.4
    mQ7
    Klenow exo-0.6
    DNA10
    total20µlx


    Water bath: 30min -- 37’C
    • Zymo purification -- elute in 10μl EB



  4. Adapter ligation
    First mix DNA with adapters, let stand for ~5min, then add buffer with ligase mix

    DNA10
    Adapter mix (for fragment library)2
    Quick ligase buffer 2x12.5
    Quick ligase1
    total25.5µlx


    Water bath: 30min -- 20’C
    • Zymo purification -- elute in 10μl EB

    You can stop here, freeze samples in LN, put them to -20°C. Reasonable to start RT-PCR and leave in ON



    [править] RT-PCR

    To check number of cycles and quality

    x
    Sybr green buffer2µl
    dNTPs2µl
    MgCl22µl
    ≠601 10µM 0,8µl
    ≠603 10µM0,8µl
    DNA (unamp lib)1µl-
    mQ11,2µl
    Immolase0,2µl
    total20µl19µl mix + 1µl DNA
    +positive control
    +negative controlmQ H2O


    ca. 2h


    Check if melting curve shows two peeks, if not stop here!


    sample12345678
    cycles
    -2 cycles for LS-PCR (9µl DNA template)



  5. Amplification

    MM 2x12.5
    PCR 1.10.5
    PCR2.10.5
    mQ1.5
    DNA10
    total25µl


    In PCR machine: (tanja_lib_ampl)

    [ 1] 98°C     30:00
    [ 2] 98°C     10:00
    [ 3] 65°C     30:00
    [ 4] 72°C     45:00
    [ 5] go to 2, ## times
    [ 6] 72°C     5:00
    



  6. Gel electrophoresis
    Prepare :
    25µl PCR mix + 10µl 4xloading buffer
    Load on 2%LRA gel; 140V; ~1hour

  7. Cut out fragments 150-350bp (not lower then 150bp!!!); 350-600bp – reserve band
  8. QiaQuick purification – elute in 2x16 µl EB
  9. Measure concentration on Qubit: 2µl in 200µl
    Further usually done by the Illumina sequencing person (Asja)
  10. Prepare 10nM dilution of the library in LSB (0.1% Tween20 in EB)
  11. Perform RT-PCR
  12. check if melting curve shows two peeks, if not stop here!


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