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==Background== * 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 [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2014-2-3]] * 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)== * 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 {| class="wikitable" |- style="font-size:12pt" align="center" valign="bottom" | width="43" height="15" | Sample | width="108" | Sample conc. (ng/ul) | width="114" | Volume for 400ng (ul) | width="114" | H2O (ul) | width="128" | Actual sample left |- style="font-size:12pt" align="center" valign="bottom" | height="15" | BR_1 | align="center" | 21.7 | align="center" | 18.43 | align="center" | 91.57 | Less than 10 ul |- style="font-size:12pt" align="center" valign="bottom" | height="15" | BR_2 | align="center" | 54 | align="center" | 7.41 | align="center" | 102.59 | OK |- style="font-size:12pt" align="center" valign="bottom" | height="15" | BR_3 | align="center" | 57.3 | align="center" | 6.98 | align="center" | 103.02 | OK |- style="font-size:12pt" align="center" valign="bottom" | height="15" | BR_4 | align="center" | 71.9 | align="center" | 5.56 | align="center" | 104.44 | OK |- style="font-size:12pt" align="center" valign="bottom" | height="15" | BR_5 | align="center" | 39.5 | align="center" | 10.13 | align="center" | 99.87 | Less than 10.13 but close |- style="font-size:12pt" align="center" valign="bottom" | height="15" | BR_6 | align="center" | 29.4 | align="center" | 13.61 | align="center" | 96.39 | Less than 10 ul |- style="font-size:12pt" align="center" valign="bottom" | height="15" | BR_7 | align="center" | 33.7 | align="center" | 11.87 | align="center" | 98.13 | Less than 10 ul |- style="font-size:12pt" align="center" valign="bottom" | height="15" | BR_8 | align="center" | 38.3 | align="center" | 10.44 | align="center" | 99.56 | Less than 10 ul |- style="font-size:12pt" align="center" valign="bottom" | height="15" | BR_9 | align="center" | 32.1 | align="center" | 12.46 | align="center" | 97.54 | Less than 10 ul |- style="font-size:12pt" align="center" valign="bottom" | height="15" | BR_10 | align="center" | 24 | align="center" | 16.67 | align="center" | 93.33 | Close to none remaining |- style="font-size:12pt" align="center" valign="bottom" | height="15" | BR_11 | align="center" | 40.3 | align="center" | 9.93 | align="center" | 100.07 | Less than 9.93 ul |- style="font-size:12pt" align="center" valign="bottom" | height="15" | BR_12 | align="center" | 46.3 | align="center" | 8.64 | align="center" | 101.36 | OK |- style="font-size:12pt" align="center" valign="bottom" | height="15" | BR_13 | align="center" | 27.8 | align="center" | 14.39 | align="center" | 95.61 | None Remaining |- style="font-size:12pt" align="center" valign="bottom" | height="15" | BR_14 | align="center" | 29.5 | align="center" | 13.56 | align="center" | 96.44 | Less than 10 ul |- style="font-size:12pt" align="center" valign="bottom" | height="15" | BR_15 | align="center" | 35.2 | align="center" | 11.36 | align="center" | 98.64 | Less than 10 ul |- style="font-size:12pt" align="center" valign="bottom" | height="15" | BR_16 | align="center" | 39 | align="center" | 10.26 | align="center" | 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.<span style="background-color:#FFFF00">BR_X|Sheared|400bp</span>->Box(<span style="background-color:#0f0">Alan 2015/6/3 Dr. Rana WGBS</span>) ==End Repairing and A-Tailing (2015-06-10)== {| class="wikitable" class="wikitable" class="wikitable" class="wikitable" class="wikitable" class="wikitable" class="wikitable" class="wikitable" class="wikitable" |- style="background-color:#BFBFBF;font-size:12pt" align="center" valign="bottom" | width="354" height="15" | Component | width="99" | Volume (uL) | width="180" | Master Mix (uL) |- style="font-size:12pt" align="center" valign="bottom" | height="15" | Fragment avg 400bp, double-stranded DNA | align="center" | 50 | Do not add |- style="font-size:12pt" align="center" valign="bottom" | height="15" | End Repair & A-Tailing Buffer | align="center" | 7 | align="center" | 130.9 |- style="font-size:12pt" align="center" valign="bottom" | height="15" | End Repair & A-Tailing Enzyme Mix | align="center" | 3 | align="center" | 56.1 |- style="font-size:12pt" align="center" valign="bottom" | height="15" | Total Volume | align="center" | 60 | align="center" | 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. {| class="wikitable" class="wikitable" class="wikitable" class="wikitable" class="wikitable" class="wikitable" |- style="background-color:#BFBFBF;font-size:12pt" align="center" | width="219" height="15" | Step | width="99" | Temp | width="107" | Time |- style="font-size:12pt" align="center" | height="15" | End Repair | 20 °C | 30 min |- style="font-size:12pt" align="center" | height="15" | A-Tailing | 65 °C | 30 min |- style="font-size:12pt" align="center" | height="15" | HOLD | 12 °C | ∞ |} * Proceed immediately to the next step ==Adapter Ligation (2015-06-10)== * Assemble each Adapter Ligation reaction as follows: {| 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="background-color:#BFBFBF;font-size:12pt" align="center" | width="236" height="16" | Component | width="348" | Volume (μl) | width="284" | MasterMix (μl) |- style="font-size:12pt" align="center" | height="16" | End Repair & A-Tailing reaction product | align="center" | 60 | Do not add |- style="font-size:12pt" align="center" | height="16" | PCR-grade water | align="center" | 9 | align="center" | 168.3 |- style="font-size:12pt" align="center" | height="16" | Ligation Buffer | align="center" | 30 | align="center" | 561 |- style="font-size:12pt" align="center" | height="16" | DNA Ligase | align="center" | 10 | align="center" | 187 |- style="font-size:12pt" align="center" | height="16" | Adapter stock | align="center" | 1 | align="center" | 18.7 |- style="font-size:12pt" align="center" | height="16" | Total volume | align="center" | 110 | align="center" | 935 |} * Incubate at 20 °C for 15 min. Proceed immediately to the next step. ==Beads Purification (2015-06-10)== * 0.8X beads purification and elute with 25ul ddh2o. * <span style="background-color:#FF0000">BR_X|Methylated|Adapter</span>->Box(<span style="background-color:#0f0">Alan 2015/6/3 Dr. Rana WGBS</span>) ==qPCR to check End Repair, A-tailing, and adapter ligation works (2015-06-11)== {| 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="151" height="15" | Components | width="31" | 1rxn | width="63" | 8.5 rxn mix |- style="font-size:12pt" align="center" valign="bottom" | height="15" | Adapter ligated DNA | align="center" | 2 | Do not Add |- style="font-size:12pt" align="center" valign="bottom" | height="15" | 2.5uM PCR_F+PCR_R N2 IndX | align="center" | 2 | Do not Add |- style="font-size:12pt" align="center" valign="bottom" | height="15" | 2x KAPA SYBG MM | align="center" | 12.5 | align="center" | 233.75 |- style="font-size:12pt" align="center" valign="bottom" | height="15" | H2O | align="center" | 8.5 | align="center" | 158.95 |- style="font-size:12pt" align="center" valign="bottom" | height="15" | Total | align="center" | 25 | align="center" | 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. * <span style="background-color:#00FFFF">BR_X|No Meth|PCR</span>->Box(<span style="background-color:#0f0">Alan 2015/6/3 Dr. Rana WGBS</span>) ==Bisulfite Conversion with Zymo Lighting Kit (2015-07-10)== * 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)== {| class="wikitable" class="wikitable" |- style="font-size:12pt" align="center" valign="bottom" | width="203" height="15" | Components | width="29" | 1rxn | width="61" | 17 rxn mix | width="103" | Final Concentration |- style="font-size:12pt" align="center" valign="bottom" | height="15" | Adapter ligated Bisulfite Converted DNA | align="center" | 3 | Do not add | n/a |- style="font-size:12pt" align="center" valign="bottom" | height="15" | 5uM PCR_F | align="center" | 2 | align="center" | 35.7 | 200nM |- style="font-size:12pt" align="center" valign="bottom" | height="15" | 5uM PCR_R.IndN2 | align="center" | 2 | Do not add | 200nM |- style="font-size:12pt" align="center" valign="bottom" | height="15" | 2x KAPA SYBG MM | align="center" | 25 | align="center" | 446.25 | 1X |- style="font-size:12pt" align="center" valign="bottom" | height="15" | H2O | align="center" | 18 | align="center" | 321.3 | n/a |- style="font-size:12pt" align="center" valign="bottom" | height="15" | Total | align="center" | 50 | align="center" | 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)== * 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== * 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== * 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
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