Noi/NOTES/2014-7-22
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Preparation of low-input RRBS sequencing libraries with MspI/TaqI double-digestion on normal plasma DNA[edit]
- Continued from [[1]]
- I will add experimental details.
3) Methylated adaptor ligation[edit]
#strip/tube | Code | TruSeq Index |
#1_1 | NC-P-12 | 1 |
#1_2 | NC-P-13 | 2 |
#1_3 | NC-P-14 | 4 |
#1_4 | NC-P-15 | 5 |
#1_5 | NC-P-16 | 6 |
#1_6 | NC-P-17 | 7 |
#1_7 | NC-P-18 | 8 |
#1_8 | NC-P-19 | 10 |
#2_1 | NC-P-20 | 13 |
#2_2 | NC-P-21 | 14 |
#2_3 | NC-P-22 | 15 |
#2_4 | NC-P-23 | 16 |
#2_5 | NC-P-24 | 27 |
#2_6 | NC-P-25 | 18 |
#2_7 | NC-P-26 | 19 |
#2_8 | NC-P-27 | 20 |
#3_1 | NC-P-29 | 11 |
#3_2 | NC-P-30 | 12 |
#3_3 | NTC1 | 21 |
#3_4 | NTC2 | 22 |
1st round amplification[edit]
- Perform 1st round PCR as previously described for 1ng low-input DNA
2nd round amplification (TEST)[edit]
- Did 2nd round PCR in small volume at 6 and 8 cycles and verified in 6% TBE gel as in previous experiment
File:ZhangLab 2 2014-07-24 16hr 21min dRRBS 6x gel1.jpg File:ZhangLab 2 2014-07-24 16hr 21min dRRBS 6x gel2.jpg
File:ZhangLab 2 2014-07-24 16hr 21min dRRBS 8x gel1.jpg File:ZhangLab 2 2014-07-24 16hr 21min dRRBS 8x gel2.jpg
2nd round amplification in a large volume[edit]
- I did 2nd PCR in larger volume (50ul X 2)
- 6 cycles for the samples with high intensity in TEST PCR and 7X for those samples with very low signal
- Purified 2nd PCR 90ul with 1X AMPure beads and eluted with 55ul EB buffer
- PAGE quantification in 6% TBE gel, volume 5ul
PAGE quantification results[edit]
- Gel1: Sample NC-P-12 - 21 (10 samples)
- Gel 2: Sample NC-P-22-30 (8 samples)
File:ZhangLab 2 2014-07-25 17hr 03min PQ dRRBS NC-P 12-21.jpg File:ZhangLab 2 2014-07-25 17hr 03min PQ dRRBS NC-P 22-30.jpg
Sample | TruSeq Index# | Index | Conc. (ng/ul) | Amount in 50ul (ng) | Volume for 30ng (ul) |
NC-P-12 | 1 | ATCACG | 4.54 | 226.89 | 6.61 |
NC-P-13 | 2 | CGATGT | 1.47 | 73.62 | 20.38 |
NC-P-14 | 4 | TGACCA | 1.51 | 75.31 | 19.92 |
NC-P-15 | 5 | ACAGTG | 3.09 | 154.27 | 9.72 |
NC-P-16 | 6 | GCCAAT | 6.55 | 327.58 | 4.58 |
NC-P-17 | 7 | CAGATC | 5.16 | 258.00 | 5.81 |
NC-P-18 | 8 | ACTTGA | 6.11 | 305.33 | 4.91 |
NC-P-19 | 10 | TAGCTT | 1.56 | 78.08 | 19.21 |
NC-P-20 | 13 | AGTCAA | 0.99 | 49.31 | 30.42 |
NC-P-21 | 14 | AGTTCC | 0.81 | 40.52 | 37.02 |
NC-P-22 | 15 | ATGTCA | 2.15 | 107.38 | 13.97 |
NC-P-23 | 16 | CCGTCC | 1.05 | 52.30 | 28.68 |
NC-P-24 | 27 | ATTCCT | 0.71 | 35.67 | 42.05 |
NC-P-25 | 18 | GTCCGC | 2.35 | 117.44 | 12.77 |
NC-P-26 | 19 | GTGAAA | 3.44 | 172.07 | 8.72 |
NC-P-27 | 20 | GTGGCC | 5.73 | 286.31 | 5.24 |
NC-P-29 | 11 | GGCTAC | 2.47 | 123.62 | 12.13 |
NC-P-30 | 12 | CTTGTA | 6.18 | 309.05 | 4.85 |
PAGE size selection[edit]
- Size-select at the range between 160-420bp in 2 of 5-well 6% TBE gel
File:ZhangLab 2 2014-08-25 18hr 39min Size-select dRRBS NC-P 12-30.jpg File:ZhangLab 2 2014-08-25 18hr 39min Size-select dRRBS NC-P 12-30 gel2.jpg
Sequencing linrary ID: NP_dRRBS_NC-P-12-30_Jul22
Quantification by qPCR: 3.47nM based on the average size ~290bp (between 160-420bp)