AlanFung:LabNotes/Sequencing/2009-12-1

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Construction of CV-iB libraries with the USER/S1 protocol[edit]

PCR with dUTP[edit]

  • Mix CV-iB #A and #C at 3:1 ratio. 10ul A + 1ul C.
  • Mix CV-iB #B, #D, #E at 2:1:1 ratio. 4ul B + 4ul D + 3ul E.
                                  x2
    DNA                    10ul
    2X Taq Master mix:    200ul
    1mM dUTP:               8ul
    100uM AmpF6.3NH2:     0.8ul
    100uM AmpR6.3NH2:     0.8ul
    H2O                   181ul
    94C 2min -> 8x (94C 30sec -> 60C 30sec -> 72C 30sec) -> 72C 3min.
    Purify each amplicon with two Qiaquick columns.
    Elute w/ 32ul EB buffer in each tube.

USER digestion[edit]

    DNA  60ul
    USER  6ul
    37C 1 hour

S1 Nuclease digestion[edit]

S1 Digestion
10X S1 Nuclease Buffer 8
DNA after USER Digestion 66
S1 Nuclease (10U/ul) 2
ddH2O 4
  • 37C 10mins
  • Minelute purification. Elute w/16uH2O x2 for each set
  • Nanodrop
A,C:20.7ng/ul*32ul=662.4ng
B,D,E:32.1ng/ul*32ul=1027.2ng
  • Fragmentation and end-polishing
  • Size Selection
  • Ligation
  • PCR of sequencing Library
  • QPCR quantification


Fragmentation and end-polishing (Make blunt ends with 5'P)[edit]

  • Make up to 85ul with dh2o (53ul each)
  • End-Repair Reactions
Fragmented DNA 85ul
10X End Repair Bufer 10ul
End Repair Enzyme Mix 5ul
  • Incubate tubes at RT for 30minutes.
  • Perform a Qiaquick purification and elute with 40ul EB buffer.
Nanodrop
A,C: 12.1ng/ul*40ul=484ng
B,D,E: 19.1ng/ul*40ul=764ng
  • NOTE: For blunt-end ligation, the A-Tailing reaction should be skipped. Doing TA ligation is preferable since it eliminates the chance of getting chimeric reads.
  • A-Tailing Reactions
Blunet-end DNA 37ul
10X dA-Tailing Reaction Buffer 5ul
Klenow Fragment (3'-5' exo-) 3ul
H2O 5ul

Incubated at 37C for 30min, purified with Qiaquick columns, eluted with 22 ul EB.

  • Measure concentration with nanodrop
A,C: 14.4ng/ul*22ul=316.8ng
B,D,E: 24.7ng/ul*22ul=543.4ng

Size selection using Invitrogen 2% SizeSelect gel[edit]

  • Fill any unused well with 30ul EB Buffer
  • Mix 15ul EB buffer with 0.5ul ladder in a 0.2ml tube
  • Load 20u end-polished DNA sample into two lanes(use 3 or more lanes if the starting DNA is more than 1ug)
  • Run the SizeSelect program for 12~14 min. Pause when the 125bp band just move into the middle collection well
  • Use pipette to extract all solution in each of the sample well, and quickly refill the wells with 20ul EB.
  • Resume the electrophoresis for additional 15sec, extract DNA and refill the wells with 20ul EB. *Resume the electrophoresis for additional 15sec, extract DNA (all the extracted DNA from the same sample are mixed in a tube)
  • Refill the wells with 20ul EB, and run the electrophoresis for additional 3 minutes. Take a picture of the gel to document the extracted fragment sizes.

File:ZhangLab 2 2009-12-01 12hr 31min.jpg

  • Concentrate the DNA with a SpeedVac (no heat) to ~36ul (takes about 1hour).

Ligation. Blunt-end and TA ligations use the same protocol but slightly different adaptor sequences.[edit]

  • Set up ligation reaction. Note that the ATP in the Quick Ligase buffer hydrolyzes very quickly after several rounds of freeze/thaw cycles, so it’s a good idea to make small aliquots of a fresh tube of Quick Ligase Buffer, and use one small aliquot each time.
ul
End-repaired & size selected DNA 36
40uM adaptor2 2
5X Quick Ligase Buffer 10
Quick Ligase 2
  • Incubate at room temperature for 15 minutes, purified with MinElute columns, eluted with 23ul EB.
Nanodrop
A,C: 15.9ng/ul*23ul=365.7ng
B,D,E: 15.6ng/ul*23ul=358.8ng

PCR of sequencing library[edit]

Solexa_PCR_up: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT

AmpR6.3Sol: CAAGCAGAAGACGGCATACGAGCTCTTCGGAACGATGAGCCTCCAAC

AmpF6.3rSol: CAAGCAGAAGACGGCATACGAGCTCTTCCAGATGTTATCGAGGTCCGA

  • prepare 2 master mix tube
' F R
10uM Solexa_PCR_up 4.4 4.4
10uM AmpR6.3Sol 0 4.4
10uM AmpF6.3Sol 4.4 0
2X Phusion 110 110
50X SYBR Green I 0.88 0.88
H2O 79.2 79.2
Ligation products 10 10
10uM solexa PCR up 2 2
10uM AmpR6.3Sol 2 -
10uM AmpF6.3Sol - 2
2X Phusion 50 50
50X SYBR Green I 0.4 0.4
H2O 36 36
  • 90ul mix per well
  • Setup


PCR program: 98 °C 30sec -> 8 cycles of (98°C 10sec -> 60°C 20 sec -> 72°C 15sec) -> 72°C 3min ->15°C hold.

  • TBE Gel verification (10well)
  • 10ul h2o+4.5ul 6x loading dye+0.5ul 25bp ladder
  • 10ul h2o+3ul 6X loading dye+2ul sample

File:ZhangLab 2 2009-12-01 15hr 00min.jpg File:ZhangLab 2 2009-12-01 15hr 27min.jpg

  • Mix the amplicons with two sets of primers
  • Purified with Minelute columns, elute with 13ul EB buffer

QPCR Quantification[edit]

Setup
Stock Phi-X (1nM)   CV-iB1 (1/10)   CV-F1 (1/10)      CV-iB-B,D,E (1/10)
Stock Phi-X (1nM)   CV-iB1 (1/10)   CV-F1 (1/10)      CV-iB-B,D,E (1/10)
Stock Phi-X (1nM)   CV-iB1 (1/10)   CV-F1 (1/10)      CV-iB-B,D,E (1/10)
Stock Phi-X (1nM)   CV-iB1 (1/10)   CV-F1 (1/10)      CV-iB-B,D,E (1/10)
Amp Phi-X (1nM)     CV-iF1 (1/10)   CV-iB-A,C (1/10)  PGP8A
Amp Phi-X (1nM)     CV-iF1 (1/10)   CV-iB-A,C (1/10)  PGP8A
Amp Phi-X (1nM)     CV-iF1 (1/10)   CV-iB-A,C (1/10)  PGP8A
Amp Phi-X (1nM)     CV-iF1 (1/10)   CV-iB-A,C (1/10)  PGP8A
X16 X1.1
DNA 2.000
Syb_FP5 (100uM) 0.200 3.200 3.520
Syb_RP7 (100uM) 0.200 3.200 3.520
2X Phusion 25.000 400.000 440.000
50X SYBR Green I 0.200 3.200 3.520
H20 24.000 384.000 422.400
872.960
  • Dilute sample, 1ul in 9ul ddh2o
  • mix 2ul 1/10 sample with 98ul mix
  • aliquot 24ul to each well
98C 30 sec -> 20 cycles of (98C 10sec->64C 20sec->72C 20sec)->72C 5 min
Result
PhiX 10.749 Cycle Difference 2^ x10 (nM)
CV-iB1 7.410 3.339 10.119 101.190
CV-iF1 7.664 3.085 8.486 84.855
CV-F1 7.320 3.429 10.770 107.704
CV-iB (A,C) 6.445 4.304 19.753 197.530
CV-iB (B,D,E) 6.459 4.290 19.562 195.622
PGP8A 6.636 4.113 17.304
Dilute to 10nM Sample (ul) ddH2O
CV-iB1 5.000 45.595
CV-iF1 5.000 37.428
CV-F1 5.000 48.852
CV-iB (A,C) 5.000 93.765
CV-iB (B,D,E) 5.000 92.811
PGP8A 5.000 3.652