Kun:LabNotes/CpgSeq/2008-3-18
Exp. 1 Cpg capture with a new batch of bisulfite treated Jurkat DNA from 03/17/08
Sigma x2 Zymo x 2 Template 7 3 10X buffer(fresh) 1 1 Cpg28k probe(218nM) 2 2 H2O 0 4 Tubes: A: Sigma, dNTP 10nM B: Sigma, dNTP 50uM C: Zymo, dNTP 10nM D: Zyno, dNTP 50uM
Exon capture control
GM20431 131ng/ul 7 (10/12/2006) 10X buffer(fresh) 1 E55kv1 probe(220nM) 2 (01/12/2008) H2O 0
94C 10min -> 60C 10min -> add 1ul NSL mix (Stoffel 2U/ul, Ampligase 0.5U/ul, dNTP 10nM or 50uM) -> 60C 1h -> 45 cycles of (94C 1min -> 60C 1h) -> 60C hold -> 37C 1min -> add 2ul Exo I&III, 37C 2h -> 94C 5min -> 4C hold.
PCR
Template 12ul 2X iProof Mastermix 100ul 100uM AmpF6.2 0.8ul 100uM AmpR6.2 0.8ul 50X SYBG I 0.8ul H2O 85ul Split into two tubes. 98C 30S -> (98C 10S -> 58C 20S -> 72C 20S) x 15 -> 72C 5 min -> 15C hold.
File:2008-03-21-Cpg-E55kv1-captured-PCR.png A: Sigma, dNTP 10nM B: Sigma, dNTP 50uM C: Zymo, dNTP 10nM D: Zymo, dNTP 50uM E: E55kv1, dNTP 10nM F: E55kv1, dNTP 50uM
To test whether some of the high MW bands are concatemers, I did Alu I digestion on the MinElute purified amplicons. 8ul DNA + 1ul 10X NEBuffer2 + 1ul Alu I -> 37C 30min -> 75C 10min. File:2008-03-21-capured-amplicon-AluI-cut.png
PAGE purification of amplicon D, F, and Cosmic C(03/17/08)
File:2008-03-21-PAGE.png Resuspended the pellets in 15ul ddH2O.
Blunt end cloning
PAGE purified amplicons: 3.5ul 10X ligation buffer: 0.5ul Zero Blunt vector: 0.5ul T4 DNA ligase: 0.5ul 16C 1h -> 4C overnight.
Colony PCR & sequencing
I pick 16 colonies from the CpG plate, 8 from the Cosmic plate and 8 from the E55kv1 plate.
PCR:
2X TaqMaster mix 15ul 10uM M13 F/R primer mix 0.6ul H2O 14.4ul 94C 2min -> 35 cycles of (94C 30sec -> 52C 30sec -> 72C 30sec) -> 72C 3min
Check with Argarose gel, 4ul amplicons per lane, the ladder is Invitrogen Low Mass ladder (4ul).
File:2008-03-27-colony-PCR.png
I estimated the concentration of each band based on the intensity of the 200bp band of the ladder.
ExoSap-IT treatment: 5ul amplicons + 2ul ExoSap-IT, 37C 15min -> 80C 15min
Samples submitted for sequencing Cpg #1: Cpg A1 Cosmic #1: Cosmic A1 Cpg #2: Cpg B1 Cosmic #2: Cosmic B1 Cpg #3: Cpg C1 Cosmic #3: Cosmic C1 Cpg #4: Cpg D1 Cosmic #4: Cosmic D1 Cpg #5: Cpg E1 E55kv1 #1: E55kv1 A1 Cpg #6: Cpg F1 E55kv1 #1: E55kv1 A1 Cpg #7: Cpg H1 E55kv1 #1: E55kv1 D1 Cpg #8: Cpg B2 E55kv1 #1: E55kv1 E1
===Analysis of sequencing results:
Insert(incl. H1&H2) Target Note Cosmic #1: 235bp FRMD4A exon 1 mismatch in H2 Cosmic #2: 65bp, contains AP1V6 and AP2V2 sequences, seems to due to chimerism in probe prep Cosmic #3: 226bp RIMS2 exon 100% match Cosmic #4: Similar to #2, also has amplification adaptors
Cpg #1
The capturing sequences were mapped to the probe targeting the CpG island at chr22:38387887-38388790. The insert is 192bp, same as the expected size.
I did blast pair-wise alignment between the insert and the targeted genomic sequence.
Query 34 CGTCGTTTTAGTTGGAATAGTTTTAAGTATAAGTCGTCGTCGGTGGAGTATGAGTTTTTG 93
||||| | || ||||| || | ||| | ||| || |||||| |||| |||||| ||
Sbjct 187 CGTCGCTCCAGCTGGAACAGCCTCAAGCACAAGCCGCCGTCGGCGGAGCATGAGTCCCTG 246
Query 94 TTTTTTGAGGAGCGCGGCGGCGGCGTTCGGGTTTGCGAGGTTGTCGCGGACGAGGGGTCG 153
| | || ||||||||||||||||| ||||| |||||||||| ||||||||||||| ||
Sbjct 247 CTCTCTGCGGAGCGCGGCGGCGGCGCCCGGGTCTGCGAGGTTGCCGCGGACGAGGGGCCG 306
Query 154 TCGTGGGTCGTATTTTTGTATATTTTATACGTTTATTACGTTTATTATGGGTTTTATTTG 213
|| ||| || | || | | | ||| | || || || | ||| || ||
Sbjct 307 CCGCGGGCCGCACCCCTGCACACCCCACACGCCCACCACATTCATCACGGGCCCCATCTG 366
Query 214 GTGTATCGTTATCGTTATTATCGTCGGACGTTGTTTTTCGATAATAGGGATTTGGTGGAT 273
| | | || | || | | || |||||| ||| |||| || ||||| | ||||||
Sbjct 367 GCGCACCGCCACCGCCACCACCGCCGGACGCTGTCCCTCGACAACAGGGACTCGGTGGAC 426
Query 274 TTGGTCG 280
||| ||
Sbjct 427 CTGGCCG 433