Noi/NOTES/2014-12-11: Difference between revisions
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= RRBS library prep, mouse | = RRBS library prep, mouse hippocampus gDNA (Zhou's lab) = | ||
[[noi:DMR220k_LabNotes#2014|'''Back to calendar''']]<br> | [[noi:DMR220k_LabNotes#2014|'''Back to calendar''']]<br> | ||
* 2014-12-01: Received 9 mouse hippocamplus gDNA from Zhou's lab | * 2014-12-01: Received 9 mouse hippocamplus gDNA from Zhou's lab | ||
Line 232: | Line 232: | ||
=== TEST PCR === | === TEST PCR === | ||
:- Prepare PCR in total 20ul reaction with 4 ul BIS-DNA | :- Prepare PCR in total 20ul reaction with 4 ul BIS-DNA | ||
{| {{table}} border = 1 | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Conc''' | |||
| align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Final conc.''' | |||
| align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''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|500px]] | |||
* 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| 500px]] | |||
{| {{table}} border = 1 | |||
| align="center" style="width:100px;background:#f0f0f0;"|'''Sample IDs''' | |||
| align="center" style="width:100px;background:#f0f0f0;"|'''TruSeq Index''' | |||
| align="center" style="width:100px;background:#f0f0f0;"|'''Seq (RC)''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Con. (ng/ul)''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''Yield in 28ul''' | |||
| align="center" style="width:80px;background:#f0f0f0;"|'''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 | |||
|} |
Latest revision as of 11:56, 24 December 2014
RRBS library prep, mouse hippocampus gDNA (Zhou's lab)[edit]
- 2014-12-01: Received 9 mouse hippocamplus gDNA from Zhou's lab
N.D. measurement by Orit[edit]
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[edit]
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[edit]
- 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[edit]
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[edit]
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[edit]
- 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[edit]
- - 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[edit]
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[edit]
- - 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[edit]
- Performed bisulfite conversion using the same procedure following manufacturer's instruction EZ DNA Methylation-Lightning™ Kit and elute with 34ul Elution buffer.
- - 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[edit]
TEST PCR[edit]
- - 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 |