Noi/NOTES/2014-6-28

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Preparation of RRBS sequencing libraries of solid tumor tissue sample from colon and lung cancer patients

Calendar

Sample info

Project ID Zhang lab ID Conc. (ng/ul)
UCSD-006-1 6-T-1 43.40
UCSD-006-2 6-T-2 31.60
UCSD-006-3 6-T-3 31.50
UCSD-006-4 6-T-4 41.70
UCSD-006-5 6-T-5 30.50
UCSD-007-1 7-T-1 30.70
UCSD-007-2 7-T-2 44.60
UCSD-007-3 7-T-3 35.90
UCSD-007-4 7-T-4 33.80
UCSD-007-5 7-T-5 20.40
  • X-Y_Z:
    • X = project ID number. 4 = pancreas, 6 = colon, 7 = lung
    • Y = sample type. T = tissue, P = plasma, S = serum
    • Z = sample ID

1) MspI digestion

  • Conditions: Digest 100ng of DNA with 20U of MspI in 30ul reaction at 37C O/N (normally 3h) and heat inactivate at 65C 20min
  • For 100ng DNA input, I used 2ul (20U) of MspI.
  • I spike in 1% of unmeth lambda DNA (1ng)
  • I actually start MspI at late night of 2014-06-27, so I incubated it O/N as it doesn't hurt to incubate DNA with MspI longer than 3h.
  • I do two replicates for each sample


MspI digestion set up

1.1) Colon samples, UCSD-006

# Sample Conc. (ng/ul) Volume for 100ng (ul) H2O to 10ul MspI Mix Total Code
1 6-T-1.1 43.40 2.30 7.70 20.00 30.00 6T-1.1
2 6-T-1.2 43.40 2.30 7.70 20.00 30.00 6T-1.2
3 6-T-2.1 31.60 3.16 6.84 20.00 30.00 6T-2.1
4 6-T-2.2 31.60 3.16 6.84 20.00 30.00 6T-2.2
5 6-T-3.1 31.50 3.17 6.83 20.00 30.00 6T-3.1
6 6-T-3.2 31.50 3.17 6.83 20.00 30.00 6T-3.2
7 6-T-4.1 41.70 2.40 7.60 20.00 30.00 6T-4.1
8 6-T-4.2 41.70 2.40 7.60 20.00 30.00 6T-4.2
9 6-T-5.1 30.50 3.28 6.72 20.00 30.00 6T-5.1
10 6-T-5.2 30.50 3.28 6.72 20.00 30.00 6T-5.2
11 6-T-NTC 0.00 0.00 10.00 20.00 30.00 6T-NTC

1.2) Lung sample, UCSD-007

# Sample Conc. (ng/ul) Volume for 100ng (ul) H2O to 10ul MspI Mix Total Code
1 7-T-1.1 30.70 3.26 6.74 20.00 30.00 7T-1.1
2 7-T-1.2 30.70 3.26 6.74 20.00 30.00 7T-1.2
3 7-T-2.1 44.60 2.24 7.76 20.00 30.00 7T-2.1
4 7-T-2.2 44.60 2.24 7.76 20.00 30.00 7T-2.2
5 7-T-3.1 35.90 2.79 7.21 20.00 30.00 7T-3.1
6 7-T-3.2 35.90 2.79 7.21 20.00 30.00 7T-3.2
7 7-T-4.1 33.80 2.96 7.04 20.00 30.00 7T-4.1
8 7-T-4.2 33.80 2.96 7.04 20.00 30.00 7T-4.2
9 7-T-5.1 20.40 4.90 5.10 20.00 30.00 7T-5.1
10 7-T-5.2 20.40 4.90 5.10 20.00 30.00 7T-5.2
11 7-T-NTC 0.00 0.00 10.00 20.00 30.00 7T-NTC

Msp I Reaction Mix

Components 1x rxn 24 rxn Mix
MspI (10U/ul) 2.00 48.00
10x Tango Buffer 3.00 72.00
1ng/ul unmeth-lambda DNA 1.00 24.00
H2O 14.00 336.00
Total 20.00

Prep
- Aliquot MspI reaction mix to 8-tube strip. 65ul for tube #1-3, #6-8 and 44ul for tube #4-5

- Add H2O to each tube
- Add 100ng 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, O/N (~13.5h) --> 65C, 20min --> hold at 4C (set heat lid)


2) End-repair/dA-tailing

Prep
- Mix 26ul of Klenow exo- with 26ul of dA:dC:dG (stock 20mM:2mM:2mM) just right before adding to MspI digested DNA
- Aliquot Klenow, exo- and dA:dC:dG mix to 8-tube strip. 7ul for tube #1-3, #6-8 and 5ul for tube #4-6

- Add 2ul of Klenow, exo- and dA:dC:dG mix to each tube of MspI digested DNA with multichannel pipette. 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)
  • 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

Prep
- Freshly prepare 8mL of 75% EtOH by mixing 6mL of 100% EtOH with 2mL of H2O (prepare when the tubes sit on magnet)
- Aliquot 1400ul of AMPure beads in 30mL reservoir and leave at RT

- Add 64ul AMPure bead with multichannel pipette. 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

  • 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

Ligation reaction set up

Prep
- Thaw TruSeq adaptor on ice (during drying the beads, keep it on ice all the time)
- Prepare ligation reaction mix (during drying the beads)

Ligation reaction Mix

Components 1x rxn 24rxn Mix
dA-tailed DNA 20.00 0.00
10x Ligation Buffer 3.00 72.00
T4 DNA Ligase (6000U/ul) 1.00 24.00
TruSeq Adaptor DNA 1.00 24.00
H2O 5.00 120.00
Total 30.00

Prep
- Aliquot ligation reaction mix to 8-tube strip. 29ul for tube #1-3, #6-8 and 20ul for tube #4-5

- Add 1ul of non-diluted TruSeq adaptors and pipette few times to make sure adaptors were added to the reaction
Sample TruSeq Index Sample TruSeq Index
6-T-1.1 1 7-T-1.1 13
6-T-1.2 2 7-T-1.2 14
6-T-2.1 23 7-T-2.1 15
6-T-2.2 4 7-T-2.2 16
6-T-3.1 5 7-T-3.1 27
6-T-3.2 6 7-T-3.2 18
6-T-4.1 7 7-T-4.1 19
6-T-4.2 8 7-T-4.2 20
6-T-5.1 25 7-T-5.1 21
6-T-5.2 10 7-T-5.2 22
6-T-NTC 10 7-T-NTC 22
- Add 9ul of ligation reaction mix with multichannel pipette
- Spin down the tube
- Mix by gentle pulse-vortexing on PCR rack 10x
- Spin down the tube
- 16C, 24h (no heat lid. Ligation time is flexible. I usually do at least 20h at 16C)
- 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 diluted AMPure beads (60ul each) and resuspend the bead with 31ul EB buffer

2014-06-29

AMPure bead purification

Prep
- Freshly prepare 8mL of 75% EtOH by mixing 6mL of 100% EtOH with 2mL of H2O (prepare when the tubes sit on magnet)
- Aliquot 670ul of AMPure beads and 670ul of 20% PEG 8000/5M NaCl in 1.5mL tube, mix well and leave at RT (forgot to dilute, used beads directly)

- Add 60ul AMPure bead with multichannel pipette. 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.
- Wait for 2min.
- Transfer to sit on magnet for 2 min and transfer 30ul of adaptor-ligated DNA to new strip tubes for bisulfite conversion (discard the beads)
- Continue to bisulfite conversion


Bisulfite conversion

  • Performed bisulfite conversion using LifeTech MethylCode™ Bisulfite Conversion Kit and did the same procedure following manufacturer's instruction and elute with 38ul Elution buffer.

Prep

- Prepare 2 tubes of complete CT Conversion Reagent by adding 800ul H2O, 50ul Resuspension Buffer, and 300ul Dilution Buffer
- Mix well and spin down the tube
- Put on thermomixer at 37C for 15min, 1,000rpm
- Solution should be clear
- Spin down the tube
- 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 transferring 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

TEST PCR

Components Conc unit Final conc./amount unit Volume (ul) 25 rxn mix
Bis-cvt DNA 6.00 0.00
10X Reaction buffer 10 X 1 X 2.00 50.00
dNTP mix 10 mM 0.25 mM 0.50 12.50
TruS_F/R 10 uM 0.3 uM 0.60 15.00
50X SYBG 50 X 0.8 X 0.32 8.00
PfuTurbo Cx 2.5 Unit/ul 1 unit 0.40 10.00
H2O 10.18 254.50
Total 20.00
- Aliquot 14 + 6ul DNA template
- Mix well. Split 2X of 8.5ul
95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X [14X & 16X] --> 72C for 2min
  • There was error to realplex during annealing step of 15 cycles. I needed to remove samples. I loaded only 14X to save the gel and plan to loaded 16 cycles PCR if the intensity of 14X is too low.
- Add 10ul of 2X loading dye to PCR product and load 10ul PCR product/dyemix in 12% gel and run at 250 volts for 40min
Colon tumor samples
File:ZhangLab 2 2014-06-30 17hr 16min 6T 100ngRRBS TEST-14X.jpg
 
Lung tumor samples
File:ZhangLab 2 2014-06-30 17hr 16min 7T 100ngRRBS TEST-14X-e.jpgg
  • I do not know why sample 7-T_3.2 and 7-T_4.1 failed. May need to check with Dr. Zhang if he want to sequence the two duplicates or only 1 library for each sample.
  • No need to load 16 cycles PCR since 14 cycles look good and not too faint or over-amplified.
  • I need to repeat 7-T_3.2 and 7-T_4.1. I plan to repeat both of them in two duplicates.
  • Continued to prepare sequencing library by doing PCR in 100ul reaction with 30% BIS-DNA.
Components Conc unit Final conc./amount unit Volume (ul) 20.5
Bis-cvt DNA 30.00 0.00
10X Reaction buffer 10 X 1 X 10.00 205.00
dNTP mix 10 mM 0.25 mM 2.50 51.25
TruS_F/R 10 uM 0.3 uM 3.00 61.50
50X SYBG 50 X 0.8 X 1.60 32.80
PfuTurbo Cx 2.5 Unit/ul 1 unit 2.00 41.00
H2O 50.90 1043.45
Total 100.00
- 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 14 cycles --> 72C for 2min
- Stopped at 14X cycles based on TEST PCR
- I pooled the two PCR replicates (total ~90ul for each samples) and purified with 1x volume AMPure beads and eluted with 55ul EB buffer
- I did PAGE quantification by loading 3ul of bead-purified sequencing libraries
File:ZhangLab 2 2014-07-02 19hr 34min PQ 6T 100ngRRBS.jpg 

File:ZhangLab 2 2014-07-02 19hr 34min PQ 7T 100ngRRBS.jpg
  • It's so wired for sample 6T_4.2 since it looked fine in TEST PCR, but it's very faint after amplification in 50ul and bead purified.
Repeat smaple 7T_4.1 (also include 7T_4.2)
File:ZhangLab 2 2014-07-03 17hr 35min PQ repeat 7T 4.jpg
* I reloaded sample 6T_2.1 and 6T_4.2 again. To confirm that they had very low concentration.

PAGE quantification results

006-T samples

Sample Index Conc. (ng/ul) Amount in 50ul (ng)
6-T-1.1 1 6.53 326.73
6-T-1.2 2 9.91 495.56
6-T-2.1 23 2.00 100.20
6-T-2.2 4 7.14 356.87
6-T-3.1 5 10.60 530.01
6-T-3.2 6 6.70 334.87
6-T-4.1 7 6.19 309.49
6-T-4.2 8 0.71 35.55
6-T-5.1 25 4.31 215.60
6-T-5.2 10 8.26 412.83

007-T samples

  • Value of sample 7-T-4.1 is the repeated sample (will be added)
Sample Index Conc. (ng/ul) Amount in 50ul (ng)
7-T-1.1 13 5.12 255.78
7-T-1.2 14 3.65 182.39
7-T-2.1 15 4.18 208.94
7-T-2.2 16 6.55 327.39
7-T-3.1 27 2.70 135.19
7-T-3.2 18 3.72 185.97
7-T-4.1 19 9.30 465.00
7-T-4.2 20 3.59 179.72
7-T-5.1 21 5.76 288.10
7-T-5.2 22 3.82 190.84

Library poolng

006-T samples
  • Since I may need to sequence only one replicate, I combine sequencing libraries into two set; set 1 and set2. Set 1 set the first priority, so I included sample 6-T-2_2 in set one as sample 6-T-2_1had very faint signal. I included sample 6-T_2.1 in set 2

NP-RRBS-6T-set1-Jun28

Sample Index Conc. (ng/ul) Volume for 120ng (ul)
6-T_1.1 1 6.53 18.36
6-T_2.2 4 7.14 16.81
6-T-3.1 5 10.60 11.32
6-T-4.1 7 6.19 19.39
6-T-5.1 25 4.31 27.83
- Add 20ul of 6X loading dye and load 37.9ul in 3 well of 5-well 6% TBE gel

NP-RRBS-6T-set2-Jun28

  • Set 2 I include 4 libraries and do PAGE size selection of sample 6-T-4.2 separately since it had very faint smear.
Sample Index Conc. (ng/ul) Volume for 100ng (ul)
6-T-1.2 2 9.91 10.09
6-T-2.1 23 2.00 49.90
6-T-3.2 6 6.70 14.93
6-T-5.2 10 8.26 12.11
- Add 20ul of 6X loading dye and load 35.68 in 3 well of 5-well 6% TBE gel

NP-RRBS-6T-4_2-set2-Jun28

Sample Index Conc. (ng/ul) Volume (ul)
6-T-4.2 8 0.71 50.00
- Add 10ul of 6X loading dye and load 60ul in 1 well of 5-well 6% TBE gel
007-T samples

NP-RRBS-7T_set1_Jun28

Sample Index Conc. (ng/ul) Volume for 100ng (ul)
7-T-1.1 13 5.12 19.55
7-T-2.1 15 4.18 23.93
7-T-3.1 27 2.70 36.98
7-T-4.1 19 9.30 10.75
7-T-5.1 21 5.76 17.35
- Add 25ul of 6X loading dye and load 44.52ul in 3 well of 5-well 6% TBE gel

NP-RRBS-7T_set2_Jun28

Sample Index Conc. (ng/ul) Volume for 100ng (ul)
7-T-1.2 14 3.65 27.41
7-T-2.2 16 6.55 15.27
7-T-3.2 18 3.72 26.89
7-T-4.2 20 3.59 27.82
7-T-5.2 22 3.82 26.20
- Add 30ul of 6X loading dye and load 51.2ul in 3 well of 5-well 6% TBE gel