Kun:LabNotes/CpgSeq/2008-3-18

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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).

   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

Cosmic amplicons

             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
 
  Raw sequences of #2
  >Cosmic2-T7.ab1
  XXXXXXXXXXGCAGCTGAATAGGAACGATGAGCCTCCAACACCCAATCTT
  AAAGTGATTGCTCCATGTTTAACAGTGCTCTTCCAGTCTACAACAGTGCT
  CTTCCAGTCTACCTAGCCTCATGCGTATCCGATCCAGAAGGCCAGAAATG
  GCAGTGTCGGACCTCGATAACATCTGCAGCTGAATAGGAACGATGAGCCT
  CCAACACCCAATCTTAAAGTGATTGCTCCATGTTTAACAGTGCTCTTCCA
  GTCTACAACAGTGCTCTTCCAGTCTACCTAGCCTCATGCGTATCCGATCC
  AGAAGGCCAGAAATGGCAGTGTCGGACCTCGATAACATCTGCA   
  Raw sequences of #4
  >Cosmic4-T7.ab1
  XXXXXXXXXXXXTGCAGATGTTATCGAGGTCCGACAAACCAATTGCACAG
  CACACGATCGGATACGCATGAGGCTATGTAGACTGGAAGAGCACTGTTAC
  ACTAAGAGCCCACTGCCAGAGGTTATGGAGTTGGAGGCTCATCGTTCCTA
  TTCAGCTGCAGATGTATCGAGGTCCGACAAACCAATTGCACAGCACACGA
  TCGGATACGCATGAGGCTATGTAGACTGGAAGAGCACTGTTACACTAAGA
  GCCCACTGCCAGAGGTTATGGAGTTGGAGGCTCATCGTTCCTATTCAGCT
  GC

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