AlanFung:LabNotes/2015/2015-1-22
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Size Selection of LMS (MONOD V5A) BSPP capture on WGBS libraries prepared from cfDNA of normal control and cancer patient
Background
- Kun thinks the results from the experiment Noi did on Illumina’s plasma samples looks promising.
- He thinks we should repeat the same experiments on all our cancer and plasma samples.
- We should have all those WGBS libraries ready, so it would be just a BSPP capturing experiment (we might need to make more probes though).
- Kun asked Noi to help me identify the reagents and design a capturing experiment on all WGBS libraries
- Noi said she had already finished the BSPP capture on WGBS libraries and she will need my help to pool the libraries and do size selection
- Noi will update her wiki so I can follow her work
- Noi used 600ng of WGBS libraries for capture instead of the 400ng for illumina.
- She reduced the probe to target ratio from 200:1 to 125:1due to limited amount of probes.
- She also used MONOD V5A probe set (LMS cluster) and the rest of the protocol are pretty much the same.
- She quantified the library concentration by PAGE quantification, there seems to be a little high variation between different gels
- She asked me to do size selection of 6 pools (8 individual libraries each) of sequencing libraries as noted in her wiki.
- I will have to cut the get in the range between 300-350bp (Do not of over 400bp) since she cropped the images to show me.
- There will probably be high background over 400bp, and that is probably ssDNA form when she did more PCR cycles (should have done 1-2 cycle less)
- Use one 5-well TBE gel per one pool of libraries
- The individual sequencing libraries are in the PCR rack on the top shelf of -20C freezer as shown in the wiki.
- Noi already filled in the sequencing spreadsheet
- For Illumina experiment, Noi suggested Li to only pool sequencing libraries and re-purified with 0.7% AMPure beads to concentrate the libraries and remove the remaining primer dimers.
- However, for Li libraries, I got low background of a smear over 400bp.
Part I: Gel Size Selection
Step 1. Pool libraries for size selection
Tube ID: NP-BSPP_V5A_NC.p1-Dec16 | |||
Samples Name | Sample ID | Concentration (ng/ul) | Volume for 70ng (ul) |
NP-BSPP-V5A-Dec16-NC-1_41 | NC-1 | 5.23 | 13.39 |
NP-BSPP-V5A-Dec16-NC-2_42 | NC-2 | 5.42 | 12.92 |
NP-BSPP-V5A-Dec16-NC-3_43 | NC-3 | 9.44 | 7.42 |
NP-BSPP-V5A-Dec16-NC-5_44 | NC-5 | 4.74 | 14.77 |
NP-BSPP-V5A-Dec16-NC-6_45 | NC-6 | 5.54 | 12.64 |
NP-BSPP-V5A-Dec16-NC-7_46 | NC-7 | 4.44 | 15.76 |
NP-BSPP-V5A-Dec16-NC-8_47 | NC-8 | 4.01 | 17.46 |
NP-BSPP-V5A-Dec16-NC-9_48 | NC-9 | 5.49 | 12.76 |
Tube ID: NP-BSPP_V5A_NC.p2-Dec16 | |||
Samples Name | Sample ID | Concentration (ng/ul) | Volume for 70ng (ul) |
NP-BSPP-V5A-Dec16-NC-12_49 | NC-12 | 2.41 | 29.09 |
NP-BSPP-V5A-Dec16-NC-13_50 | NC-13 | 2.14 | 32.66 |
NP-BSPP-V5A-Dec16-NC-14_51 | NC-14 | 2.7 | 25.91 |
NP-BSPP-V5A-Dec16-NC-15_52 | NC-15 | 2.65 | 26.45 |
NP-BSPP-V5A-Dec16-NC-16_53 | NC-16 | 2.6 | 26.95 |
NP-BSPP-V5A-Dec16-NC-17_54 | NC-17 | 3.37 | 20.8 |
NP-BSPP-V5A-Dec16-NC-18_55 | NC-18 | 2.26 | 31.01 |
NP-BSPP-V5A-Dec16-NC-19_56 | NC-19 | 3.55 | 19.72 |
Tube ID: NP-BSPP_V5A_NC.p3-Dec16 | |||
Samples Name | Sample ID | Concentration (ng/ul) | Volume for 70ng (ul) |
NP-BSPP-V5A-Dec16-NC-20_57 | NC-20 | 5.5 | 18.17 |
NP-BSPP-V5A-Dec16-NC-21_58 | NC-21 | 6.45 | 15.52 |
NP-BSPP-V5A-Dec16-NC-22_59 | NC-22 | 6.02 | 16.61 |
NP-BSPP-V5A-Dec16-NC-23_60 | NC-23 | 6.1 | 16.38 |
NP-BSPP-V5A-Dec16-NC-25_61 | NC-25 | 5.68 | 17.61 |
NP-BSPP-V5A-Dec16-NC-27_62 | NC-27 | 5.85 | 17.08 |
NP-BSPP-V5A-Dec16-NC-29_63 | NC-29 | 5.82 | 17.18 |
NP-BSPP-V5A-Dec16-NC-30_64 | NC-30 | 7.19 | 13.9 |
Tube ID: NP-BSPP_V5A_PCP-Dec16 | |||
Samples Name | Sample ID | Concentration (ng/ul) | Volume for 70ng (ul) |
NP-BSPP-V5A-Dec16-PCP-2_65 | PCP-2 | 3.06 | 22.9 |
NP-BSPP-V5A-Dec16-PCP-3_66 | PCP-3 | 2.32 | 30.13 |
NP-BSPP-V5A-Dec16-PCP-4_67 | PCP-4 | 2.55 | 27.46 |
NP-BSPP-V5A-Dec16-PCP-5_68 | PCP-5 | 3.56 | 19.65 |
NP-BSPP-V5A-Dec16-PCP-6_69 | PCP-6 | 1.99 | 35.16 |
NP-BSPP-V5A-Dec16-PCP-7_70 | PCP-7 | 2.25 | 31.16 |
NP-BSPP-V5A-Dec16-PCP-8_71 | PCP-8 | 2.07 | 33.81 |
NP-BSPP-V5A-Dec16-PCP-9_72 | PCP-9 | 4.03 | 17.36 |
Tube ID: NP-BSPP_V5A_6P-Dec16 | |||
Samples Name | Sample ID | Concentration (ng/ul) | Volume for 70ng (ul) |
NP-BSPP-V5A-Dec16-6P-1_73 | 6P-1 | 4.68 | 17.08 |
NP-BSPP-V5A-Dec16-6P-2_74 | 6P-2 | 3.99 | 20.05 |
NP-BSPP-V5A-Dec16-6P-3_75 | 6P-3 | 6.79 | 11.77 |
NP-BSPP-V5A-Dec16-6P-4_76 | 6P-4 | 4.62 | 17.31 |
NP-BSPP-V5A-Dec16-6P-5_77 | 6P-5 | 4.63 | 17.26 |
NP-BSPP-V5A-Dec16-6P-8_78 | 6P-8 | 5.58 | 14.35 |
NP-BSPP-V5A-Dec16-6P-9_79 | 6P-9 | 3.99 | 20.05 |
NP-BSPP-V5A-Dec16-6P-10_80 | 6P-10 | 4.87 | 16.42 |
Tube ID: NP-BSPP_V5A_7P-Dec16 | |||
Samples Name | Sample ID | Concentration (ng/ul) | Volume for 70ng (ul) |
NP-BSPP-V5A-Dec16-7P-1_81 | 7P-1 | 3.42 | 20.48 |
NP-BSPP-V5A-Dec16-7P-2_82 | 7P-2 | 1.6 | 43.62 |
NP-BSPP-V5A-Dec16-7P-3_83 | 7P-3 | 3.86 | 18.13 |
NP-BSPP-V5A-Dec16-7P-5_84 | 7P-5 | 3.22 | 21.74 |
NP-BSPP-V5A-Dec16-7P-6_85 | 7P-6 | 3.25 | 21.54 |
NP-BSPP-V5A-Dec16-7P-7_86 | 7P-7 | 4.18 | 16.75 |
NP-BSPP-V5A-Dec16-7P-8_87 | 7P-8 | 3.48 | 20.13 |
NP-BSPP-V5A-Dec16-7P-10_88 | 7P-10 | 3.69 | 18.98 |
Step 2. Add Gel Loading Dye and Load Gel
Sample ID | Total | 6X Dye | Sum | Per Well (4x) |
Tube ID: NP-BSPP_V5A_NC.p1-Dec16 | 107.12 | 22 | 129.12 | 32.28 |
Tube ID: NP-BSPP_V5A_NC.p2-Dec16 | 212.59 | 45 | 257.59 | 64.4 |
Tube ID: NP-BSPP_V5A_NC.p3-Dec16 | 132.44 | 30 | 162.44 | 27.07 |
Tube ID: NP-BSPP_V5A_PCP-Dec16 | 217.63 | 45 | 262.63 | 65.66 |
Tube ID: NP-BSPP_V5A_6P-Dec16 | 134.3 | 30 | 164.3 | 41.07 |
Tube ID: NP-BSPP_V5A_7P-Dec16 | 181.37 | 40 | 221.37 | 55.34 |
Step 3. Gel Running & Size Selection Conditions
- Run gel at 200V for 30mins
- Stain gel with 4uL SYBR Gold in ~100 mL of TBE in clean tray.
- Place tray on an orbital shaker for ~5 min.
- Prepare two gel shearing assemblies by placing a 0.5 mL tube within another 1.5 mL Non-Stick tube. Use G20 needle to punch a small hole at the bottom of the 0.5 mL tube. Use two assemblies for each PAGE gel.
- Cut out the correct band (300-350 bp in this case), chop the band into small slices, pool two gel slices into one gel shearing assembly.
- Change gloves and plastic wrap when size selecting a new gel.
- Centrifuge at top speed > 12000 rpm for 2 min to sheer the gel, remove the 0.5 mL tube.
- Add 400 uL clean 1x TE buffer in the 1.5 mL tube. Tape the tubes on a vortexer, place the vortexer in 37C incubator, shake the tubes at low speed for 1 hour. Alternatively, can keep the tubes in 4C fridge overnight.
- Spin down all tubes at 13,000rpm for 2min
- In the PCR hood, transfer the liquid layer of the gel mix to Nanosep columns, spin for 3 min at top speed (15,000 rpm), and then transfer supernatant to 1.5mL tube.
Step 4. Precipitation
Turn on centrifuge let it cool down to 4C
- Add 0.1x total volume of 3M sodium acetate and 2.5x total volume 100% ethanol.
- Add 1uL Glycoblue vortex and spin down so that one can precipitate better and see the DNA.
- Freeze in -80C for 20 min, spin at 10,000 rpm for 20 min at 4C, discard supernatant with pipette.
- Add 500 uL of 75% ethanol to wash DNA, spin at 10,000 rpm for 5 min at 4C, discard supernatant and aspirate with (P1000 pipette) let dry for 5 mins spindown ad aspirate with P200 pipette.
- Let dry in hood ~ 10 min.
- Add 20 uL of water(totally) to the tube to resolve the DNA in, leave at least 15 min.
Store at 4C if needed, spin down before proceeding