Revision as of 15:40, 16 December 2014 by >Noi
RRBS library prep, mouse Mouse hippocampus gDNA (Zhou's lab)
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- 2014-12-01: Received 9 mouse hippocamplus gDNA from Zhou's lab
N.D. measurement by Orit
Sample ID
|
ng/ul
|
A260
|
A280
|
260/280
|
260/230
|
C1 |
113.72 |
2.274 |
1.199 |
1.9 |
2.32
|
C2 |
84.47 |
1.689 |
0.919 |
1.84 |
2.19
|
C3 |
109.47 |
2.189 |
1.207 |
1.81 |
1.79
|
C4 |
116.84 |
2.337 |
1.273 |
1.84 |
2.23
|
H1 |
115.85 |
2.317 |
1.285 |
1.8 |
2.14
|
H2 |
107.32 |
2.146 |
1.161 |
1.85 |
2.11
|
H3 |
106.12 |
2.122 |
1.14 |
1.86 |
2.11
|
H3 |
90.76 |
1.815 |
0.997 |
1.82 |
2.17
|
H4 |
145.22 |
2.904 |
1.68 |
1.73 |
1.29
|
H5 |
96.04 |
1.921 |
1.042 |
1.84 |
2.2
|
- I re-quantified concentration by Qubit dsDNA HS assay using 1ul for quantification
Qubit dsDNA HS quantification result
Sample
|
Conc. in the Qubit
|
Unit
|
uL used
|
Dilution
|
Conc. (ng/ul)
|
HC-C1 |
459 |
ng/mL |
1 |
200 |
91.80
|
HC-C2 |
382 |
ng/mL |
1 |
200 |
76.40
|
HC-C3 |
510 |
ng/mL |
1 |
200 |
100.00
|
HC-C4 |
510 |
ng/mL |
1 |
200 |
100.00
|
HC-H1 |
487 |
ng/mL |
1 |
200 |
97.40
|
HC-H2 |
495 |
ng/mL |
1 |
200 |
99.10
|
HC-H3 |
395 |
ng/mL |
1 |
200 |
79.00
|
HC-H4 |
493 |
ng/mL |
1 |
200 |
98.60
|
HC-H5 |
428 |
ng/mL |
1 |
200 |
85.60
|
1) MspI digestion
- I will use 500ng for RRBS library prep and spike in with 1ng of unmethylated lambda DNA for monitoring bisulfite conversion rate
- Conditions: Digest 500ng of DNA with 20U of MspI in 30ul reaction at 37C 3h and heat inactivate at 65C 20min
MspI digestion set up
Sample ID
|
Conc. (ng/ul)
|
Volume for 500ng (ul)
|
Sample code
|
H2O
|
MspI Mix
|
Total (ul)
|
HC-C1 |
91.80 |
5.45 |
C1 |
4.55 |
20.00 |
30.00
|
HC-C2 |
76.40 |
6.54 |
C2 |
3.46 |
20.00 |
30.00
|
HC-C3 |
100.00 |
5.00 |
C3 |
5.00 |
20.00 |
30.00
|
HC-C4 |
100.00 |
5.00 |
C4 |
5.00 |
20.00 |
30.00
|
HC-H1 |
97.40 |
5.13 |
H1 |
4.87 |
20.00 |
30.00
|
HC-H2 |
99.10 |
5.05 |
H2 |
4.95 |
20.00 |
30.00
|
HC-H3 |
79.00 |
6.33 |
H3 |
3.67 |
20.00 |
30.00
|
HC-H4 |
98.60 |
5.07 |
H4 |
4.93 |
20.00 |
30.00
|
HC-H5 |
85.60 |
5.84 |
H5 |
4.16 |
20.00 |
30.00
|
NTC |
0.00 |
0.00 |
NTC |
10.00 |
20.00 |
30.00
|
Msp I reaction Mix
Components
|
1 rxn
|
10.5 rxn mix
|
MspI (10U/ul) |
2.00 |
21.00
|
10X Tango Buffer |
3.00 |
31.50
|
1ng/ul unmeth-lambda DNA |
1.00 |
10.50
|
H2O |
14.00 |
147.00
|
Total |
20.00 |
|
- - Add H2O to each tube
- - Add 500ng of DNA to each tube
- - Add 20ul 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, 3h --> 65C, 20min --> hold at 4C (set heat lid)
2) End-repair/dA-tailing
- After MspI, I took 2ul of MspI-digested DNA for gel verification (equal to DNA ~33.33ng). Total volume left is 28ul
- Mix 13ul of 5U/ul Klenow exo- (Thermoscientific) with 13ul of 20:2:2mM dAdCdG
- Add 2ul of Klenow exo-/dAdCdG mix to each tube. Total volume is 30ul
- - Spin down the tubes
- - Mix by gentle pulse-vortexing on PCR rack 10x
- - Spin down the tubes
- - 30C, 20min --> 37C, 20min --> hold at 4C (no heat lid)
- - 75C, 10min --> hold at 4C (heat lid on)
- - Perform AMPure bead purification with 2x volume AMPure beads
AMPure bead purification
- - Add 60ul AMPure bead. Mix by pipetting 10x
- - Wait for 15min
- - Transfer to sit on magnet for 5min
- - Wash twice with 200ul freshly prepared 80% EtOH
- - After adding 80% EtOH (1st time), wait for 30sec before pipetting up & down 5x
- - Remove all 80% EtOH
- - Dry the bead for 15min
- - Resuspend with 20 EB Buffer (keep the beads in the tubes!)
Methylated adaptor ligation
Components
|
1x rxn
|
11rxn Mix
|
dA-tailed DNA |
20.00 |
0.00
|
10x Ligation Buffer |
3.00 |
33.00
|
T4 DNA Ligase (6000U/ul) |
1.00 |
11.00
|
TruSeq Adaptor (15uM) |
1.50 |
0.00
|
H2O |
4.50 |
49.50
|
Total |
30.00 |
|
- - Add 1.5ul of 15uM (non-diluted adaptor) TruSeq adaptor to each tube
Sample
|
Truseq Index
|
HC-C1 |
1
|
HC-C2 |
2
|
HC-C3 |
3
|
HC-C4 |
4
|
HC-H1 |
5
|
HC-H2 |
6
|
HC-H3 |
7
|
HC-H4 |
8
|
HC-H5 |
9
|
NTC |
10
|
- Final conc. of adaptor in 30ul lgation reaction is 750nM
- - Add 8.5ul of ligation reaction mix with adaptors
- - Spin down the tube
- - Mix by gentle pulse-vortexing on PCR rack 10x
- - Spin down the tube
- - 16C, 24h (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 21ul EB buffer
AMPure bead purification
- - Add 60ul AMPure bead. Mix by pipetting 10x
- - Wait for 15min
- - Transfer to sit on magnet for 5min
- - Wash twice with 200ul freshly prepared 80% EtOH
- - After adding 80% EtOH (1st time), wait for 30sec before pipetting up & down 5x
- - Remove all 80% EtOH
- - Dry the bead for 15min
- - Resuspend with 21 EB Buffer
- - Wait for 2min.
- - Transfer to sit on magnet for 2 min and transfer 20ul of adaptor-ligated DNA to new strip tubes (discard the beads)
- - Continue to bisulfite conversion
Bisulfite conversion
- - Add 130ul complete Lightning 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 (Magprep, Heated Lid)
- - 98°C for 10 minutes (DNA denaturation)
- - 54°C for 1 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)
- - Wait for 18min
- - 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 36ul 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
Amplification
TEST PCR
- - Prepare PCR in total 20ul reaction with 4 ul BIS-DNA
Components
|
Conc
|
unit
|
Final conc.
|
unit
|
Volume (ul)
|
10.5 rxn mix
|
Bis-cvt DNA |
|
|
|
|
3.00 |
0.00
|
2X KAPA HiFi Uracil HotStart ReadyMix |
2 |
X |
1 |
X |
5.00 |
52.50
|
TruS_F/R |
10 |
uM |
0.3 |
uM |
0.30 |
3.15
|
50X SYBG |
50 |
X |
0.4 |
X |
0.08 |
0.84
|
H2O |
|
|
|
|
1.62 |
17.01
|
Total |
|
|
|
|
10.00 |
|
- - Aliquot 3.5ul + 1.5ul BIS-DNA
- - 98C for 45sec --> [98C for 15sec -> 60C for 30sec -> 72C for 30sec] X 13X --> 72C for 1min
- - Verify all PCR product in 6% TBE gel
File:ZhangLab 2 2014-12-12 20hr 24min RRBS-DZ-5ul.jpg
- It looked like the pattern of size distribution was different from previous experiment as the size seemed to be more larger than sequencing library prepared from human tumor DNA.
- I continued to amplified the rest of BIS-DNA in 100ul reaction with cycle number 14 cycles for sample C1, C2, H1, H3, 15 cycles for samples C4, H4, H5 and 16 cycles for sample C3
- - Purified with 1X AMPure bead, elute with 32ul EB buffer
- - PAGE quantify by loading 4ul
File:ZhangLab 2 2014-12-15 19hr 52min PQ SeqLib-4ul.jpg
Sample IDs
|
TruSeq Index
|
Seq (RC)
|
Con. (ng/ul)
|
Yield in 28ul
|
Volume for 75ng (ul)
|
HC-C1 |
1.00 |
ATCACG |
6.66 |
186.53 |
11.26
|
HC-C2 |
2.00 |
CGATGT |
7.07 |
198.07 |
10.60
|
HC-C3 |
3.00 |
TTAGGC |
3.16 |
88.38 |
23.76
|
HC-C4 |
4.00 |
TGACCA |
7.95 |
222.62 |
9.43
|
HC-H1 |
5.00 |
ACAGTG |
8.53 |
238.81 |
8.79
|
HC-H2 |
6.00 |
GCCAAT |
7.86 |
220.06 |
9.54
|
HC-H3 |
7.00 |
CAGATC |
9.33 |
261.24 |
8.04
|
HC-H4 |
8.00 |
ACTTGA |
7.78 |
217.90 |
9.64
|
HC-H5 |
9.00 |
GATCAG |
6.64 |
186.03 |
11.29
|