Noi/NOTES/2014-7-22

From ZhangLabWiki
Jump to navigation Jump to search

Preparation of low-input RRBS sequencing libraries with MspI/TaqI double-digestion on normal plasma DNA[edit]

Calendar

  • 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)