Noi/NOTES/2014-12-11

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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
  • 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