Sam:LabNotes/Human/2009-12-17
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Solexa sequencing library construction - human single cell MDA amplicons (1,10,1+10hr incubation)
Background
- The MDA amplicons were EtOH purified and followed by NlaIII digestion (12-16-'09). Total voulmn is 20uL.
- Due to the loading capacity of TBE gel. Only 10uL was used for library construction. The starting amounts for each samples are:
- ~0.2 (ng/uL) * 8uL / 20uL x 10uL = 0.8 ng - for 1hr-incubation samples (1-1, 1-2)
- ~50 (ng/uL) * 8uL / 20uL x 10uL = 200 ng - for 10 and 10+1 hr-incubation samples (10-1, 10-2, 11-1, 11-2)
TBE-PAGE size selection
- TBE-PAGE condition: 5-well TBU. Run at 200V, 30min
- Sample: 10uL sample + 5uL H2O + 5uL 6X loading dye
- Ladder: 0.5uL 25bp ladder + 15uL H2O + 5uL 6X loading dye
- Stained with 3uL SYBR-Gold in 0.5X TBE for 5min
- Cut the size 250+/-50bp (200bp~300bp)
File:Sam121609-precut-sample 1-2-3.JPG File:Sam121609-postcut-sample 1-2-3.JPG File:Sam121609-precut-sample 4-5-6.JPG File:Sam121609-postcut-sample 4-5-6-postcut.JPG Left: Pre-cutting Right: Post-cutting Sample annotation: #1-1(1hr-T3), #1-2(1hr-T6), #10-1(10hr T2), #10-2(10hr-T5), #11-1(1+10hr T3), #11-2(1+10hr T6)
NOTE: The starting amount of DNA for sample #1-1 and #1-2 are too few to been seen on PAGE-TBE gel.
- I only perform the DNA recovery for 10-1,10-2, 11-1 and 11-2 samples.
Size selected DNA recovery from TBE-PAGE
- 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 for 1hr on the shaker. shaking at lowest speed.
- Pellet the gel-TE buffer mixture at 14000 rpm at RT for 1.5min. Transfer the supernatant (~300uL) on 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 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.
Blunting
- End-it Kit (EPICENTRE)
- Set reactions on cold block
1rxn 4.5rxn H2O 8.0 - DNA 20.0 - 10X blunting buf. 4.0 18.0 dNTP mix 4.0 18.0 Blunting Enzyme 4.0 18.0 ------------------------------------ 40.0 54.0 54/4.5=12 --- DNA 28uL Add 1uL of DNA pol.I (E.coli) for nicks filling on each reaction - (These are NlaIII digested samples)
- 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 Pre-wrmed EB.
A-tailing
- Exo-Minus Klenow DNA polymerase(EPICENTRE)
- Set reactions on cold block
- Prepare the fresh dilution of 100mM dATP to 1mM dATP
1rxn 4.5rxns(EPI) DNA 30 - 10X buf. 4 18 (10X Knenow buf.) 1mM dATP 6 27 Klenow enzyme 2 9 ------------------------------------------ 40 54/4.5=12 - DNA 30uL
- Mix the reaction by vortexing 5sec. Incubate at 37C for 30min at Thermocycler.
- 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
- T4 ligation kit (Enzymatic)
- Set reactions on cold block
- Adaptor:Insert calculation
- Assume that only 25% DNA left after all of previous steps. 200ng*25% =50ng
- DNA before ligation: 90ng of 250bp
- 50/(660x250)x10^6/1000 = 0.303uM --- Insert. Using 10:1~50:1 (Adaptor: Insert)will be 3.03uM ~ 15.15uM Y-adaptor needed.
- I decided to use 2uL of 20uM Y-adaptor which is even higher than 50:1 rule.
4.5rxn A-tailed DNA 11 - 2X rapid liagas buf. 15 67.5 20uM Y-adaptor 2 9.0 T4 ligase 2 9.0 --------------------------------- 30 85.5/4.5 = 19uL --- DNA 11 uL
- 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
- 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 H2O + 10uL 2X TBU-loading dye
- Heat denaturing: 75C 15min -> 1min on cool block
- Loading: Low mass ladder -> No.10 -> No.12 -> 25bp ladder (Left to right)
- Stained with 3uL SYBR-Gold in 0.5X TBE for 5min
File:Sam121809-ligation size selection -precut.jpg File:Sam121809-ligation size selection -postcut.jpg Left: Pre-cutting Right: Post-cutting
Size selected DNA recovery from TBU-PAGE
- 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 for 1hr on the shaker. shaking at lowest speed.
- Pellet the gel-TE buffer mixture at 14000 rpm at RT for 1.5min. Transfer the supernatant on 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 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
- Only use half of amount(10uL) of ligation product for qPCR amplification.
- Solexa lib. templates:
- Left to right: 10-1, 10-2, 11-1, 11-2
- Primers: From Dr.Zhang (Yellow-cap)
- Working solution: 10uM
Master mix 4.5 rxn H2O 31.6 142.2 Template 10.0 - PCR_F (10uM) 4.0 18.0 PCR_R (10uM) 4.0 18.0 SYBR 50X 0.4 1.8 2X Fusion enzyme mix(Enzymatics) 50.0 225.0 ----------------------------------------------- 100.0 405.0/4.5=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:Sam122109-libamp-step5.jpg File:Sam122109-libamp-step6 and above.jpg The reaction were terminated at: 10-2, 11-1(8th cycle), 10-1(9th cycle), 11-2(10th cycle)
TBE-PAGE validation
- Sample well: 3uL sample + 3uL 6X loading dye + 3uL 0.5X TBE
- Ladder: 0.5uL low mass ladder + 3uL 6X loading dye + 6uL 0.5X TBE
- 10-well TBE gel. Run at 200V for 30min.
File:Sam122209-qPCR amplib.jpg The sample 10-1 and 11-2 seem a little bit over amplified. The expected size is accurate on 400bp (300bp sample + 100bp adaptor)
Product purification
- Purify the amplified library with QIAquick column.
- Take 20uL sample and eluted with 15uL EB.
- Gave the purified libraries to Alice for Solexa sequencing.
Lib. name Sample Sequencing tag ---------------------------------------------------------- sam003 10-1 s_1, HC-MDA-hCell-Dec17-sam003 sam004 10-2 s_2, HC-MDA-hCell-Dec17-sam004 sam005 11-1 s_3, HC-MDA-hCell-Dec17-sam005 sam006 11-2 s_7, HC-MDA-hCell-Dec17-sam006
- Stored the rest of unpurified samples in -20C.
NOTE:
- For the qPCR amplification. I also tried the newly synthesized primer(PCR_F and PCR-R). Although the templates were succesuflly amplified, the amplification curves were not consistent with the TBE-PAGE results.
- I am wondering if my qPCR didn't perform well on the final vortexing and spining step. There may have some reaction sticked to the tube cap.
- The current results were completed using the original qPCR primer (From Dr. Zhang) by a repeat qPCR reaction.
- I will need to try the qPCR amplification on those newly synthesized primer again.