AlanFung:LabNotes/2015/2015-6-3: Difference between revisions

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* Place the column into a 1.5 ml microcentrifuge tube and add 10 μl of M-Elution Buffer directly to the column matrix. Centrifuge for 30 seconds at full speed to elute the DNA.
* Place the column into a 1.5 ml microcentrifuge tube and add 10 μl of M-Elution Buffer directly to the column matrix. Centrifuge for 30 seconds at full speed to elute the DNA.
* The DNA is ready for immediate analysis or can be stored at or below -20°C for later use. -80C for long term storage.
* The DNA is ready for immediate analysis or can be stored at or below -20°C for later use. -80C for long term storage.
==PCR Amplification and Barcoding (2015-07-10)==
==PCR Amplification and Barcoding (2015-08-03)==
{| class="wikitable" class="wikitable"
{| class="wikitable" class="wikitable"
|- style="font-size:12pt" align="center" valign="bottom"
|- style="font-size:12pt" align="center" valign="bottom"
Line 344: Line 344:


|}
|}
  98C 2min --> (98C 15sec -> 64C 20sec -> 72C 30sec) x 12 cycles --> 72C 3min -->  15C Hold
  98C 2min --> (98C 15sec -> 64C 20sec -> 72C 30sec(Plate Read) -> 72C 10sec) x 19 cycles --> 72C 3min -->  15C Hold
* Monitor curves and extract before libraries reach plateau.
==Beads Purification (2015-08-10)==
* 0.8X beads purification
* Elute with 20ul ddh2o.
 
==Pool Libraries==
{| class="wikitable" class="wikitable" class="wikitable" class="wikitable" class="wikitable" class="wikitable" class="wikitable" class="wikitable" class="wikitable" class="wikitable" class="wikitable" class="wikitable" class="wikitable" class="wikitable" class="wikitable"
|- style="font-size:12pt" align="center" valign="bottom"
| width="65" height="15" | Sample
| width="85" | Nanodrop ng/ul
| width="65" | Vol ul
| width="65" | Amount ng
| width="65" | Total Vol ul
| width="74" | Total Amount
| width="65" | Conc. Ng/ul
 
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | BR_1
| align="center" | 6
| align="center" | 2.22
| align="center" | 13.3
| align="center" | 23.18
| align="center" | 199.5
| align="center" | 8.61
 
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | BR_2
| align="center" | 5.8
| align="center" | 2.29
| align="center" | 13.3
| NA
| NA
| NA
 
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | BR_3
| align="center" | 5.1
| align="center" | 2.61
| align="center" | 13.3
| NA
| NA
| NA
 
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | BR_4
| align="center" | 7.5
| align="center" | 1.77
| align="center" | 13.3
| NA
| NA
| NA
 
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | BR_5
| align="center" | 8.1
| align="center" | 1.64
| align="center" | 13.3
| NA
| NA
| NA
 
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | BR_6
| align="center" | 11.8
| align="center" | 1.13
| align="center" | 13.3
| NA
| NA
| NA
 
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | BR_7
| align="center" | 9.8
| align="center" | 1.36
| align="center" | 13.3
| NA
| NA
| NA
 
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | BR_8
| align="center" | 8.7
| align="center" | 1.53
| align="center" | 13.3
| NA
| NA
| NA
 
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | BR_9
| align="center" | 10.2
| align="center" | 1.30
| align="center" | 13.3
| NA
| NA
| NA
 
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | BR_10
| align="center" | 11.1
| align="center" | 1.20
| align="center" | 13.3
| NA
| NA
| NA
 
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | BR_11
| align="center" | 10.2
| align="center" | 1.30
| align="center" | 13.3
| NA
| NA
| NA
 
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | BR_14
| align="center" | 11.5
| align="center" | 1.16
| align="center" | 13.3
| NA
| NA
| NA
 
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | BR_15
| align="center" | 13.3
| align="center" | 1.00
| align="center" | 13.3
| NA
| NA
| NA
 
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | BR_16
| align="center" | 9.5
| align="center" | 1.40
| align="center" | 13.3
| NA
| NA
| NA
 
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | BR_12
| align="center" | 10.5
| align="center" | 1.27
| align="center" | 13.3
| NA
| NA
| NA
 
|}
 
==Library quantification==
[[File:08122015_Rana_Lib.jpg|400px]]


==Process sample #13==
==Process sample #13==
Line 351: Line 506:
* Transfer to tube.
* Transfer to tube.
* Quantify using nanodrop, the read out is negative. We will have to move on without this sample.
* Quantify using nanodrop, the read out is negative. We will have to move on without this sample.
==IGM Sequencing==
* Index
{| class="wikitable"
|- style="font-size:12pt"  valign="bottom"
| width="122" height="15" | Academic Senate Grant
| width="51" | PST084H
| align="center" width="47" | $10,000
|- style="font-size:12pt"  valign="bottom"
| height="15" | K01 Grant
| PST4913
| align="center" | $6,500
|}
* Highseq high-throughput PE 100bp
* Library Info
{| class="wikitable" class="wikitable" class="wikitable"
|- style="font-size:12pt" align="center" valign="bottom"
| width="136" height="15" | HL-Rana-WGBS_Pool-Jun3
| width="85" | 34.67nM
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | Read 1 Primer
| illumina
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | Index Primer
| N2IndSeq
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | Read 2 Primer
| N2RevSeq2
|}
* Loading Concentration recommended by illumina
{| class="wikitable" class="wikitable"
|- style="font-size:12pt" align="center" valign="bottom"
| width="122" height="15" | Library
| width="62" | Instrument
| width="65" | Loading pM
| width="120" | Cluster Density K/mm2
| width="65" | -20% pM
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | Our Library
| Miseq V3
| align="center" | 16
| align="center" | 1400
| NA
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | PhiX
| Miseq V3
| align="center" | 20
| align="center" | 1400
| align="center" | 16
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | PhiX
| Hiseq V3
| align="center" | 12
| 750-850
| align="center" | 9.6
|- style="font-size:12pt" align="center" valign="bottom"
| height="15" | PhiX
| Hiseq V4
| align="center" | 18
| 950-1050
| align="center" | 14.4
|}
* Loading Concentration recommended by IGM

Latest revision as of 21:10, 24 August 2015

Background[edit]

  • Noi made a batch of WGBS libraries on the whole blood DNA from Dr. Rana’s lab.
  • Dr. Rana has obtained some sequencing fund, and would like to get them sequenced.
  • Noi's experiment notes on wiki [[1]]
  • Noi's wiki page shows that the library preparation failed and didn't have time to repeat experiment
  • Dinh found the gDNA samples from Dr. Rana in Noi's box.
  • From the labels, Noi got about 1 ug of the samples and only used 200 ng, we should have 800ng for library prep.
  • Noi confirmed that the experiment failed for the first round of experiment and she did try a second time and still the result was not very good
  • She got very faint smear or barely see a smear in many samples by amplifying about 16-18 cycles, which is very unlikely for DNA input ~100ng
  • Noi doesn't know why this is the case, may be it is the expired beads used for purification but I will give a a try.
  • I will repeat the library prep follow the exact protocol that you did with the tumor WGBS libraries. Using new beads and switch to Kapa Hyper for library preparation.

Covaris gDNA shearing (2015-06-09)[edit]

  • Called IGM to reserve the Covaris Shearing Machine.
  • I will shear 400ng of samples in case we need to repeat experiment again.
  • Transfer 400ng of samples and make up to 110ul with ddh2o
Sample Sample conc. (ng/ul) Volume for 400ng (ul) H2O (ul) Actual sample left
BR_1 21.7 18.43 91.57 Less than 10 ul
BR_2 54 7.41 102.59 OK
BR_3 57.3 6.98 103.02 OK
BR_4 71.9 5.56 104.44 OK
BR_5 39.5 10.13 99.87 Less than 10.13 but close
BR_6 29.4 13.61 96.39 Less than 10 ul
BR_7 33.7 11.87 98.13 Less than 10 ul
BR_8 38.3 10.44 99.56 Less than 10 ul
BR_9 32.1 12.46 97.54 Less than 10 ul
BR_10 24 16.67 93.33 Close to none remaining
BR_11 40.3 9.93 100.07 Less than 9.93 ul
BR_12 46.3 8.64 101.36 OK
BR_13 27.8 14.39 95.61 None Remaining
BR_14 29.5 13.56 96.44 Less than 10 ul
BR_15 35.2 11.36 98.64 Less than 10 ul
BR_16 39 10.26 99.74 Less than 10 ul
  • Transfer to Covaris tube at IGM
  • Perform shearing
Followed up the set up for Covaris shearing from previous experiment
Target BP 400
Tube Micro tube 
Duty Factor 10%
Peak Incident Power (w) 140
Cycle per Burst 200
Time (secs) 55
Volume 50ul
Temperature (c) 7 
Intensifier Yes
  • Transfer sheared samples to non-stick 1.5mL tubes and label.BR_X|Sheared|400bp->Box(Alan 2015/6/3 Dr. Rana WGBS)

End Repairing and A-Tailing (2015-06-10)[edit]

Component Volume (uL) Master Mix (uL)
Fragment avg 400bp, double-stranded DNA 50 Do not add
End Repair & A-Tailing Buffer 7 130.9
End Repair & A-Tailing Enzyme Mix 3 56.1
Total Volume 60 187
  • Premix buffer and enzyme mastermix in a tube, aliquot 10ul to each well in a strip tube.
  • Add 50ul samples to each well and mix by pipetting.
Step Temp Time
End Repair 20 °C 30 min
A-Tailing 65 °C 30 min
HOLD 12 °C
  • Proceed immediately to the next step

Adapter Ligation (2015-06-10)[edit]

  • Assemble each Adapter Ligation reaction as follows:
Component Volume (μl) MasterMix (μl)
End Repair & A-Tailing reaction product 60 Do not add
PCR-grade water 9 168.3
Ligation Buffer 30 561
DNA Ligase 10 187
Adapter stock 1 18.7
Total volume 110 935
  • Incubate at 20 °C for 15 min. Proceed immediately to the next step.

Beads Purification (2015-06-10)[edit]

  • 0.8X beads purification and elute with 25ul ddh2o.
  • BR_X|Methylated|Adapter->Box(Alan 2015/6/3 Dr. Rana WGBS)

qPCR to check End Repair, A-tailing, and adapter ligation works (2015-06-11)[edit]

Components 1rxn 8.5 rxn mix
Adapter ligated DNA 2 Do not Add
2.5uM PCR_F+PCR_R N2 IndX 2 Do not Add
2x KAPA SYBG MM 12.5 233.75
H2O 8.5 158.95
Total 25 212.5
98C 2min --> (98C 15sec -> 64C 20sec -> 72C 30sec) x 12 cycles --> 72C 3min -->  15C Hold

File:AlanPCR12015611.jpg

  • All samples are amplified except #13.
  • BR_X|No Meth|PCR->Box(Alan 2015/6/3 Dr. Rana WGBS)

Bisulfite Conversion with Zymo Lighting Kit (2015-07-10)[edit]

  • Add 130ul lighting conversion reagent to 20ul of sample, compensate with water if less than 20ul.
  • Vortex and centrifuge.
  • (98C 8min > 54C 60min > 10C hold)
  • Add 600ul of M-Binding Buffer to column with collection tube.
  • Load the sample to column. Close the cap and mix by inverting the column several times.
  • Centrifuge at full speed for 30s. Discard flow through.
  • Add 100 μl of M-Wash Buffer to the column. Centrifuge at full speed for 30s.
  • Add 200 μl of L-Desulphonation Buffer to the column and let stand at RT for 120 mins. After the incubation, centrifuge at full speed for 30 seconds.
  • Add 200 μl of M-Wash Buffer to the column. Centrifuge at full speed for 30 seconds.
  • Repeat this wash step.
  • Place the column into a 1.5 ml microcentrifuge tube and add 10 μl of M-Elution Buffer directly to the column matrix. Centrifuge for 30 seconds at full speed to elute the DNA.
  • The DNA is ready for immediate analysis or can be stored at or below -20°C for later use. -80C for long term storage.

PCR Amplification and Barcoding (2015-08-03)[edit]

Components 1rxn 17 rxn mix Final Concentration
Adapter ligated Bisulfite Converted DNA 3 Do not add n/a
5uM PCR_F 2 35.7 200nM
5uM PCR_R.IndN2 2 Do not add 200nM
2x KAPA SYBG MM 25 446.25 1X
H2O 18 321.3 n/a
Total 50 803.25 n/a
98C 2min --> (98C 15sec -> 64C 20sec -> 72C 30sec(Plate Read) -> 72C 10sec) x 19 cycles --> 72C 3min -->  15C Hold
  • Monitor curves and extract before libraries reach plateau.

Beads Purification (2015-08-10)[edit]

  • 0.8X beads purification
  • Elute with 20ul ddh2o.

Pool Libraries[edit]

Sample Nanodrop ng/ul Vol ul Amount ng Total Vol ul Total Amount Conc. Ng/ul
BR_1 6 2.22 13.3 23.18 199.5 8.61
BR_2 5.8 2.29 13.3 NA NA NA
BR_3 5.1 2.61 13.3 NA NA NA
BR_4 7.5 1.77 13.3 NA NA NA
BR_5 8.1 1.64 13.3 NA NA NA
BR_6 11.8 1.13 13.3 NA NA NA
BR_7 9.8 1.36 13.3 NA NA NA
BR_8 8.7 1.53 13.3 NA NA NA
BR_9 10.2 1.30 13.3 NA NA NA
BR_10 11.1 1.20 13.3 NA NA NA
BR_11 10.2 1.30 13.3 NA NA NA
BR_14 11.5 1.16 13.3 NA NA NA
BR_15 13.3 1.00 13.3 NA NA NA
BR_16 9.5 1.40 13.3 NA NA NA
BR_12 10.5 1.27 13.3 NA NA NA

Library quantification[edit]

File:08122015 Rana Lib.jpg

Process sample #13[edit]

  • Dr. Rana dropped off a sample plate but she is not sure how much DNA is left in the plate.
  • Spin down the plate and take out the samples
  • Transfer to tube.
  • Quantify using nanodrop, the read out is negative. We will have to move on without this sample.

IGM Sequencing[edit]

  • Index
Academic Senate Grant PST084H $10,000
K01 Grant PST4913 $6,500
  • Highseq high-throughput PE 100bp
  • Library Info
HL-Rana-WGBS_Pool-Jun3 34.67nM
Read 1 Primer illumina
Index Primer N2IndSeq
Read 2 Primer N2RevSeq2
  • Loading Concentration recommended by illumina
Library Instrument Loading pM Cluster Density K/mm2 -20% pM
Our Library Miseq V3 16 1400 NA
PhiX Miseq V3 20 1400 16
PhiX Hiseq V3 12 750-850 9.6
PhiX Hiseq V4 18 950-1050 14.4
  • Loading Concentration recommended by IGM