Noi/NOTES/2014-6-13: Difference between revisions
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= RRBS library preparation of colon tumor tissue samples from Biochain (STD RRBS, 100ng input DNA) = | = RRBS library preparation of colon tumor tissue samples from Biochain (STD RRBS, 100ng input DNA) = | ||
[[noi:DMR220k_LabNotes|'''Calendar''']] | |||
* After I have tried STD protocol to get it worked in 12878 sample, I continued to work on CTT samples. | * After I have tried STD protocol to get it worked in 12878 sample, I continued to work on CTT samples. | ||
* The main things to concern are concentration of adaptor concentration in ligation reaction and optimization of PCR cycle number to prevent over-amplification of sequencing libraries. | * The main things to concern are concentration of adaptor concentration in ligation reaction and optimization of PCR cycle number to prevent over-amplification of sequencing libraries. | ||
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==== PAGE quantification results ==== | ==== PAGE quantification results ==== | ||
{| {{table}} class = wikitable | {| {{table}} class = wikitable | ||
| align="center" style=" | | align="center" style="background:#f0f0f0;"|'''Sample''' | ||
| align="center" style="width:80px;background:#f0f0f0;"|'''TruSeq Index''' | | align="center" style="width:80px;background:#f0f0f0;"|'''TruSeq Index''' | ||
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | ||
Line 259: | Line 259: | ||
| CTT-FFPE-100ng_2||Ind_21||1.77||79.63||19.78 | | CTT-FFPE-100ng_2||Ind_21||1.77||79.63||19.78 | ||
|} | |} | ||
:- I pooled 35ng each library based on PAGE quantification and did size selection in 2 well of 5-well | :- I pooled 35ng each library based on PAGE quantification and did size selection in 2 well of 5-well 6% TBE gel | ||
[[File:ZhangLab_2 2014-06-17 14hr 06min_100ng_CTTRRBS_PAGE-SS.jpg| 350px]] | [[File:ZhangLab_2 2014-06-17 14hr 06min_100ng_CTTRRBS_PAGE-SS.jpg| 350px]] | ||
:- I precipitated in 3tubes at -80C O/N and resuspended each tube with | :- I precipitated in 3tubes at -80C O/N and resuspended each tube with 11ul H2O --> total volume 33ul | ||
:- I took | :- I took 4ul for PAGE verification. | ||
''' | ==== PAGE verification ==== | ||
[[File:ZhangLab_2 2014-06-24 17hr 06min_RRBS_PAGE-verify.jpg| 500px]] | |||
'''Sequencing library ID: NP-RRBS-CTT-100ng, Jun13, 2014'''<br> | |||
- Length: 160-360bp<br> | - Length: 160-360bp<br> | ||
- Conc. by Qubit: XX ng/ul or XX nM | - Conc. by Qubit: XX ng/ul or XX nM |
Latest revision as of 22:29, 4 July 2014
RRBS library preparation of colon tumor tissue samples from Biochain (STD RRBS, 100ng input DNA)[edit]
- After I have tried STD protocol to get it worked in 12878 sample, I continued to work on CTT samples.
- The main things to concern are concentration of adaptor concentration in ligation reaction and optimization of PCR cycle number to prevent over-amplification of sequencing libraries.
- In this experiment, I spiked in 10pg of unmethylated lambda DNA which accounts for 0.01% of input DNA. This might be too low.
Experimental procedures[edit]
- Sample list
- CTT-frozen, 100ng, two replicates
- CTT-FFPE, 100ng, two replicates
1) MspI digestion[edit]
- Conditions: Digest 100ng of DNA with 20U of MspI in 30ul reaction at 37C 3h and heat inactivate at 65C 10min
Sample | Conc. (ng/ul) | Volume for 100ng (ul) | 10X Tango Fuffer | MspI (10U/ul) | H2O to adjust volume (ul) | 12pg/ul unmeth-lambda DNA | H2O in MspI mix(ul) | Total (ul) |
1.1 CTT-frozen | 52.30 | 1.91 | 3.00 | 2.00 | 12.26 | 0.83 | 10.00 | 30.00 |
1.2 CTT-FFPE | 64.80 | 1.54 | 3.00 | 2.00 | 12.63 | 0.83 | 10.00 | 30.00 |
1.3 NTC | 0.00 | 0.00 | 3.00 | 2.00 | 14.17 | 0.83 | 10.00 | 30.00 |
Msp I reaction Mix[edit]
Components | 1 rxn | 5.5 rxn mix |
MspI (10U/ul) | 2.00 | 11.00 |
10X Tango Buffer | 3.00 | 16.50 |
12pg/ul unmeth-lambda DNA | 0.83 | 4.57 |
H2O | 10.00 | 55.00 |
Total | 15.83 |
- - Add H2O to each tube
- - Add 100ng of DNA to each tube
- - Add 15.83ul of MspI reaction Mix to each tube and pipette few times
- - Spin down the tubes
- - Mix by gentle pulse-vortexing on PCR rack 10x
- - Spin down the tubes
- - 37C, 12h --> 65C, 20min --> hold at 4C (set heat lid)
- - Note: For this experiment, I set up MspI at night, so I incubate O/N. Longer incubation did not increase digestion that much. However, it has been reported that it does not cause star activity.
2) End-repair/dA-tailing[edit]
Prep
- Mix 6.5ul of Klenow exo- with 6.5ul of dA:dC:dG (stock 20mM:2mM:2mM) just right before adding to MspI-digested DNA
- - Add 2ul of Klwnow, exo- and dA:dC:dG mix to each tube of MspI digested DNA. Pipette few times
- - Spin down the tube
- - Mix by gentle pulse-vortexing on PCR rack 10x
- - Spin down the tube
- - 30C, 20min --> 37C, 20min --> hold at 4C (no heat lid)
- - 75C, 10min --> hold at 4C (heat lid on)
- Conditions: Add 1ul of non-diluted TruSeq adaptors (do not know actual concentration of adaptors) in 30ul ligation reaction with 6000U of T4 DNA ligase
- Before set up ligation reaction, purify end-repaired DNA with 2x volume AMPure beads (64ul each) and resuspend the bead with 20ul EB buffer
AMPure bead purification[edit]
Prep
- Freshly prepare 5mL of 75% EtOH by mixing 3.75mL of 100% EtOH with 1.25mL of H2O (prepare when the tubes sit on magnet)
- Aliquot 710ul of AMPure beads in 1.5mL tube and leave at RT
- - Add 64ul AMPure bead. Mix by pipetting 10x
- - Wait for 30min
- - Transfer to sit on magnet for 5min
- - Wash twice with 180ul freshly prepared 75% EtOH
- - After adding 75% EtOH (1st time), wait for 30sec before pipetting up & down 5x
- - Remove all 75% EtOH
- - Dry the bead for 20min
- - Resuspend with 20 EB Buffer (keep the beads in the tubes!)
3) Methylated adaptor ligation[edit]
- Conditions: Add 1ul of non-diluted TruSeq adaptors (do not know actual concentration of adaptors) in 30ul ligation reaction with 6000U of T4 DNA ligase
- Before set up ligation reaction, purify end-repaired DNA with 2x volume AMPure beads (64ul each) and resuspend the bead with 20ul EB buffer
AMPure bead purification[edit]
Prep
- Freshly prepare 5mL of 75% EtOH by mixing 3.75mL of 100% EtOH with 1.25mL of H2O (prepare when the tubes sit on magnet)
- Aliquot 325ul of AMPure beads in 1.5mL tube and leave at RT
- - Add 64ul AMPure bead. Mix by pipetting 10x
- - Wait for 30min
- - Transfer to sit on magnet for 5min
- - Wash twice with 180ul freshly prepared 75% EtOH
- - After adding 75% EtOH (1st time), wait for 30sec before pipetting up & down 5x
- - Remove all 75% EtOH
- - Dry the bead for 20min
- - Resuspend with 20 EB Buffer (keep the beads in the tubes!)
Ligation reaction Mix[edit]
Compinents | 1x rxn | 5.2x rxn |
dA-tailed DNA | 20.00 | 0.00 |
10X Ligation Buffer | 3.00 | 15.60 |
T4 DNA ligase | 1.00 | 5.20 |
H2O | 5.00 | 26.00 |
total | 29.00 |
- - Add 1ul of non-diluted TruSeq adaptors and pipette few times to make sure adaptors were added to the reaction
- - Add 9ul of ligation reaction mix
- - Spin down the tube
- - Mix by gentle pulse-vortexing on PCR rack 10x
- - Spin down the tube
- - 16C, 20h (no heat lid)
- - Mix by gentle pulse-vortexing on PCR rack 5x
- - Quickly spin down
- - Heat inactivate at 65C, 20min --> hold at 4C (heat lid on)
- Before continue to bisulfite conversion, purify adaptor-ligated DNA with 2x volume AMPure beads (60ul each) and resuspend the bead with 31ul EB buffer
Sample | TruSeq Index |
CTT-frozen-100ng_1 | Ind_18 |
CTT-frozen-100ng_2 | Ind_19 |
CTT-FFPE-100ng_1 | Ind_20 |
CTT-FFPE-100ng_2 | Ind_21 |
NTC | Ind_22 |
AMPure bead purification[edit]
Prep
- Freshly prepare 4mL of 75% EtOH by mixing 3mL of 100% EtOH with 1mL of H2O (prepare when the tubes sit on magnet)
- Aliquot 155ul of AMPure beads and 155ul of 20% PEG 8000/5M NaCl in 1.5mL tube, mix well and leave at RT
- - Add 60ul AMPure bead. Mix by pipetting 10x
- - Wait for 30min
- - Transfer to sit on magnet for 5min
- - Wash twice with 180ul freshly prepared 75% EtOH
- - After adding 75% EtOH (1st time), wait for 30sec before pipetting up & down 5x
- - Remove all 75% EtOH
- - Dry the bead for 20min
- - Resuspend with 31 EB Buffer and transfer 30ul of adaptor-ligated DNA to new strip tubes (discard the beads)
- - Continue to bisulfite conversion
4) Bisulfite conversion[edit]
- Performed bisulfite conversion using the same procedure following manufacturer's instruction [[1]] and elute with 38ul Elution buffer.
Prep
- - Mix well and spin down the tube
- - Put on thermomixer at 37C for 15min, 1000rpm
- - Solution should be clear
- - Spin down the tube
- - Note: I used CT conversion resuspended on 2014-06-11
- - Add 120ul complete CT Converson Reagent to 30ul bead-purified adaptor ligated DNA --> total volume 150ul
- - Mix by pipetting 10X with multi-channel pipette
- - Spin down the tube
- - Incubate following below program
- - 98°C for 10 minutes (DNA denaturation)
- - 64°C for 2.5 hours (Bisulfite conversion)
- - 4°C storage for up to 20 hours or continue to desulfonation
- - Add 600ul Binding Buffer to each column
- - Adjust p200 to 200ul and transfer bisulfite treated DNA to the column
- - Mix by pipetting 5x
- - Spin down column at 14,000 rpm for 30s
- - Discard spnt
- - Add 100ul Wash Buffer (Aliquot Wash Buffer in 5mL tube)
- - Spin down column at 14,000 rpm for 30s (do not discard spnt)
- - Add 200ul Desulfonation Buffer (aliquot Desulfonation Buffer in 2mL or 5mL tube)
- - Spin down column at 14,000 rpm for 30s (do not discard spnt)
- - Add 200ul Wash Buffer
- - Spin down column at 14,000 rpm for 30s
- - Discard spnt
- - Add 200ul Wash Buffer
- - Spin down column at 14,000 rpm for 3min (do not discard spnt)
- - Transfer column to prepared 1.5mL LoBind tube & make sure that column does not touch spnt in the collecting tube before transfering to new tube
- - Add 38ul of Elution Buffer (warm Elution Buffer at 60C for 10min)
- - Wait for 2min
- - Spin down column at 12,000 rpm for 1min
- - Continue to amplification (During elution step, PCR mix should be prepared)
Amplification[edit]
TEST PCR[edit]
Components | Conc | unit | Final conc. | unit | Volume (ul) | 6.5 rxn mix |
Bis-cvt DNA | 6.00 | 0.00 | ||||
10X Reaction buffer | 10 | X | 1 | X | 2.00 | 13.00 |
dNTP mix | 10 | mM | 0.25 | mM | 0.50 | 3.25 |
TruS_F/R | 10 | uM | 0.3 | uM | 0.60 | 3.90 |
PfuTurbo Cx | 2.5 | Unit/ul | 2.5 | unit | 0.40 | 2.60 |
50X SYBG | 50 | X | 0.8 | X | 0.32 | 2.08 |
H2O | 10.18 | 66.17 |
- - Aliquot 14 + 6ul DNA template
- - Mix well. Split 2X of 9ul
- 95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 18 cycles --> 72C for 2min
- - Stopped at 15X and 17X
- - Added 10ul 2X loading dye to each strip tube & mix well
- - Load 10ul PCR/dye mix in 12% gel and run at 250 volts for 40min (running in regular 6% TBE gel at 250 volts, 25 min is okay)
File:ZhangLab 2 2014-06-16 13hr 10min CTT-RRBS-test15x-17X.jpg
- - Determine # of cycles based on the intensity of the gel. For CTT-frozen samples, I would stop at 15X . For CTT-FFPE samples which have a faint smear, I would stopped at 17X.
- Then set up reaction 100ul for each samples with 30ul BIS-DNA and split into 2X of 50ul to avoid bias from amplification (Ref. split 4X of 25ul)
Components | Conc | unit | Final conc. | unit | Volume (ul) | 8.5 rxn mix |
Bis-cvt DNA | 15.00 | 0.00 | ||||
10X Reaction buffer | 10 | X | 1 | X | 5.00 | 42.50 |
dNTP mix | 10 | mM | 0.25 | mM | 1.25 | 10.63 |
TruS_F/R | 10 | uM | 0.3 | uM | 1.50 | 12.75 |
PfuTurbo Cx | 2.5 | Unit/ul | 2.5 | unit | 1.00 | 8.50 |
50X SYBG | 50 | X | 0.8 | X | 0.80 | 6.80 |
H2O | 25.45 | 216.33 |
- - I did not include NTC as it clear in TEST PCR
- - Aliquot 70 + 30ul DNA template
- - Mix well. Split 2X of 50ul (actual ~48ul)
- 95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 18 cycles --> 72C for 2min
- - Stopped at 15X for CTT-frozen and 17X for CTT-FFPE
- - I took 5ul of PCR product from each tube and mix with 5ul of 2X loading buffer to verify that PCR work (even PCR curve looked fine)
- - I pooled the two PCR replicatea (total ~87ul for each samples) and purified with 1x volume AMPure beads and eluted with 50ul EB buffer
- - I did PAGE quantification by loading 4ul of bead-purified sequencing libraries
File:ZhangLab 2 2014-06-16 17hr 52min CTT-RRBS 100ng PQ.jpg
PAGE quantification results[edit]
Sample | TruSeq Index | Conc. (ng/ul) | Amount in 45ul (ng) | Volume for 35ng (ul) |
CTT-frozen-100ng_1 | Ind_18 | 4.80 | 215.85 | 7.30 |
CTT-frozen-100ng_2 | Ind_19 | 4.64 | 208.89 | 7.54 |
CTT-FFPE-100ng_1 | Ind_20 | 1.05 | 47.31 | 33.29 |
CTT-FFPE-100ng_2 | Ind_21 | 1.77 | 79.63 | 19.78 |
- - I pooled 35ng each library based on PAGE quantification and did size selection in 2 well of 5-well 6% TBE gel
File:ZhangLab 2 2014-06-17 14hr 06min 100ng CTTRRBS PAGE-SS.jpg
- - I precipitated in 3tubes at -80C O/N and resuspended each tube with 11ul H2O --> total volume 33ul
- - I took 4ul for PAGE verification.
PAGE verification[edit]
File:ZhangLab 2 2014-06-24 17hr 06min RRBS PAGE-verify.jpg
Sequencing library ID: NP-RRBS-CTT-100ng, Jun13, 2014
- Length: 160-360bp
- Conc. by Qubit: XX ng/ul or XX nM