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
- Continued from [[1]]
- I will add experimental details.
3) Methylated adaptor ligation
#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
- Perform 1st round PCR as previously described for 1ng low-input DNA
2nd round amplification (TEST)
- Did 2nd round PCR in small volume at 6 and 8 cycles and verified in 6% TBE gel as in previous experiment
2nd round amplification in a large volume
- 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
- 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