Sam:LabNotes/Human-Chip-bisulfite-Seq/2009-11-19

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Low-input DNA Solexa seq. library construction test using dUTP-carrier[edit]

Background[edit]

  • It has been proved that dUTP-carrier can be digested by USEr enzyme and S1 endonuclease.
  • I am testing if dUTP-carrier can protect low-input DNA in library construction.
    • The low-input DNA sample were obtained from the NlaIII digested Jurket DNA (25ng/uL) from 10/23/'09 exp.

Carrier preparation[edit]

  • EtOH/NaOAC purify the dUTP-PCR product made on 11/17/'09
    • 100uL dUTP-PCR product + 250uL 100%EtOH + 10uL 3M NaOAc + 1.5uL Glycoblue -> -80C precipitation -> 750uL 75% EtOH washing
    • E1U-e: 386.52 ng/uL 1ug=2.58uL
    • E3U-e: 181.06 ng/uL 1ug=5.52uL

Sample preparation[edit]

  • NlaIII digested Jurket DNA 500ng/20uL=25ng/uL
           50ng-Carrier   100ng-Carrier   200ng-Carrier
DNA sample    2.00uL           4.00uL             8.00uL
E1U-e(1ug)    2.58             2.58               2.58 ------ 1ug dUTP-dsDNA carrier  
H2O          23.42            21.42              17.42    
--------------------------------------------------------
             28.00            28.0               28.00
           50ng-Carrier   100ng-Carrier   200ng-Carrier
DNA           2.00uL           4.00uL             8.00uL
E3U-e(1ug)    5.52             5.52               5.52 ------ 1ug dUTP-dsDNA carrier     
H2O          20.48            18.48              14.48    
--------------------------------------------------------
             28.00             28.00              28.0

Blunting[edit]

  • End-it Kit (EPICENTRE)
    • Set reactions on cold block
                      1rxn      7rxn
DNA                   28.0       -
10X blunting buf.      4.0     28.0
dNTP mix               4.0     28.0
Blunting Enzyme        4.0     28.0
------------------------------------
                      40.0     84.0   84/7=12 --- DNA 28uL
Add the DNA pol.I (E.coli) 1uL on the top of reaction
  • Mix the reaction by vortexing 5sec. Incubate at RT for 30min.
    • During the incubation, set up QIAquick washing set and thaw the reagents for the following steps (A-tailing)
  • No inactivation needed, but purify the blunted DNA with QIAquick column. Elute in 30uL EB.

NOTE: The reaction B3-2 and B3-3 maybe mis-labeled in this step.

A-tailing[edit]

  • Exo-Minus Klenow DNA polymerase(EPICENTRE)
    • Set reactions on cold block
  • Prepare the fresh dilution of 100mM dATP to 1mM dATP
                 1rxn    7rxns
DNA               30       -
10X Knenow buf.    4      28 
1mM dATP           6      42
Klenow enzyme      2      14
------------------------------------------
                  40      84/7=12 - DNA 30uL
  • Mix the reaction by vortexing 5sec. Incubate at 37C for 30min.
    • During the incubation, set up QIAquick washing set and thaw the reagents for the following steps (Ligation)
  • No inactivation needed, but purify the A-tailed DNA with QIAquick column. Elute in 12uL EB.

Ligation[edit]

  • T4 ligation kit (Enzymatic)
    • Set reactions on cold block
  • Adaptor:Insert calculation
    • Assume that 50% DNA left after all of previous steps (no sample size-selpection). (1ug + X ng)/2=500ng (X=sample amount -> ignored in the calculation)
    • DNA before ligation: 500ng of 250bp
    • 500/(660x250)x10^6/1000 = 3.03uM --- Insert. Using 10:1~50:1 (Adaptor: Insert)will be 30.3uM ~ 151.5uM Y-adaptor needed.
    • I decided to use 3uL of 20uM Y-adaptor which is in between 10:1 and 50:1 rule.
                              7rxn 
 A-tailed DNA           11      -
 2X rapid liagas buf.   15    105.0
 20uM Y-adaptor          3     21.0
 T4 ligase               2     14.0
 ----------------------------------
                        30    140.0/7 = 20uL --- DNA 10uL 

NOTE: In order to increase production, I decided to add all of the DNA (12uL) eluted from previous step.

  • Mix the reaction by vortexing 5sec. Incubate at RT for 15min.
  • No inactivation needed, but purify the ligated DNA with QIAquick column. Elute in 10uL EB.

TBU-PAGE size selection[edit]

  • TBU-PAGE condition: 5-well TBU. Run at 200V, 30min
    • Sample: 10uL of ligation reaction + 10uL 2X TBU-loading dye
    • Ladder: 0.5uL 25bp ladder + 9uL 0.5X TBE + 10uL 2X TBU-loading dye
    • Heat denaturing: 75C 15min -> on cool block (>1min)
    • Stained with 3uL SYBR-Gold in 0.5X TBE for 5min
File:Sam111909-tbu gel 1.jpg File:Sam111909-tbu gel 1-post cut.jpg
Left: Pre-cutting                                        Right: Post-cutting


File:Sam111909-tbu gel 3.jpg File:Sam111909-tbu gel 3-post cut.jpg
Left: Pre-cutting                                        Right: Post-cutting

Recovery of size selected DNA[edit]

  • Gel-cut slice was sheared by centrifuging at 14000 rpm at RT, 3min using a 22G needle punched 0.5mL tube.
  • Sheared gel was soaked in 400uL clean 1X TE and incubate at 37C shaker (>2hr or overnight). shaking at the lowest speed.
  • Pellet the gel-TE buffer mixture at 14000 rpm at RT for 1.5min. Transfer the supernatant onto a Nanosep column filter.
  • Centrifuge the Nanosep columns at 14000 rpm at RT for 1.5min. Collect the flow through.
  • Perform DNA purification with EtOH/NaOAc method.
    • Sample + 2.5V 100% EtOH + 0.1V 3M NaOAc + 1.5uL Glycoblue. Incubate at -80C for 25min (or longer)
    • Pellet the DNA precipitation by centrifuging at 4C, 10,000xg for 25min. Remove the supernatant.
    • Wash the pellet with 750uL 75% EtOH. Pellet the DNA precipitation by centrifuging at 4C, 10,000xg for 5min. Remove the supernatant.
    • Air dry the pellet in PCR hood (~5min). Elute the DNA in 20uL H2O.

qPCR amplification[edit]

  • Only use half of amount (10uL)of ligation product for qPCR amplification.
  • Solexa lib. templates:
    • Left to right: L1-1, L1-2, L1-3, L3-1, L3-2, L3-3
  • Primers - from Dr. Zhang
    • (Yellow cap, Tm=70.7, MW=17617.4) Solexa_PCR_upH (10uM)
    • (Yellow cap, Tm=72.4, MW=18710.1) Solexa_PCR_PE_lo_H (10uM)
Master mix
                                          6.5 rxn
H2O                                31.6   205.4
Template                           10.0     -
Solexa_PCR_upH (10uM)               4.0    26.0
Solexa_PCR_PE_lo_H (10uM)           4.0    26.0
SYBR 50X                            0.4     2.6
2X Fusion enzyme mix(Enzymatics)   50.0   325.0
-----------------------------------------------
                                  100.0   585.0/6.5=90 --- 10uL template
  • Using two annealing temp program for amplification
    • Use 5 cycles for the first annealing temp(64C) - step5.
    • Use 16 cycles for the second annealing temp(72C) - step9.
  • Immediately transfer 50uL of reaction into a fresh PCR tube for an extra cycle amplification in Chromo4.
  • Terminate/pause the reaction at annealing step when it's closing to saturation stage.
File:Sam112009-solexa lib qPCR step5.jpg  File:Sam112009-solexa lib qPCR step9.jpg
The reaction were terminated at the 9th and 10th cycles
File:Sam112309-low input DNA library.jpg 
Carrier size: L1(260 bp), L3(245 bp)
  • The result suggested that the library from 50ng input-DNA can barely be seen on gel. 50ng probably is the minimum of lowest input-DNA for libaray consturction.
  • A problem of undigested carrier remained in the DNA sample.
    • Based on the size it seems these carrier are also ligated with adaptors (L1: 260->360bp; L3: 245->345bp)
    • However these carriers are from know sequnce of E.coli gDNA. They could be easily removed from sequnce result computationally.