Alice:Exome/2014-1-9

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Whole genome library prep (gDNA from Dr. Linzhao Cheng Lab in Johns Hopkins University)[edit]

  • Received genomic DNA for the 6 samples of BC1 cells targeting the AAVS1 locus by either TALEN or Cas9-gRNA yesterday
  • All of the samples are ~200ng/ul in 40ul
  • Sheared at IGM facility in UCSD, program the Covaris for target size of 300 bp
List of samples:
1) BC1 p 16+14 (parental)
2) BC1 p16+21 (control)
3) BC1 AAV GFP TALEN c3 p16+21
4) BC1 AAV GFP TALEN c6 p16+21
5) BC1 AAV GFP hCas9 c4 p16+21
6) BC1 AAV GFP hCas9 c16 p16+21
  • Kapa Library construction kit for Illumina is used (KK8201)

End-repair Reactions[edit]

Fragmented DNA                             100 ul
10X End Repair Reaction Buffer              12 ul
End Repair Enzyme Mix                       5 ul
Keep the tube at ~20°C for 30 minutes.
Ampure bead purification (1.6x) and elute in 32.5ul ddH2O

Note: for blunt-end ligation, the A-Tailing reaction should be skipped. Doing TA ligation is preferable since it eliminates the
chance of getting chimeric reads.

A-Tailing reactions[edit]

Blunt-end DNA                             30 ul
10X dA-Tailing Reaction Buffer (10X)       5 ul
A Tailing Enzyme                           3 ul
H2O                                              12 ul
Incubated at 30C for 30min
Ampure bead purification (1.8x) and elute in 32.5ul ddH2O

Adaptor ligation[edit]

Prepare 30uM adaptors: 
100uM PE-t: 30ul
100uM PE-b: 30ul
10x stoffel buffer:  10ul
H2O:        30ul
94C for 3 min, and then cool down to 20C at the rate of 0.1C/sec. 
commonly used adaptors:
Blunt-end adaptors:
5’NH2- ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’OH         Solexa_1_up
3’NH2-ATGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA-5’OH            Solexa_1_lo_nop

TA adaptors (for the one adaptor protocol):
5- CAAGCAGAAGACGGCATACGAGCTCTTCCGATCT-3’OH        PE_t_adapter
3-CTTCTGCCGTATGCTCGAGAAGGCTAG-5’Phos                    t_adaptor_rc_s
  
regular Y adaptor:
PE_t_adaptor(top)              ACACTCTTTCCCTACACGACGCTCTTCCGATC*T              3'-Phosphorothioate bond    
PE_b_adaptor(bottom)           \\5phos\\GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG       5'-phosphorylation    

Note that the ATP in the Quick Ligase buffer hydrolyzes very quickly after several rounds of freeze/thaw cycles, 
so it’s a good idea to make small aliquots of a fresh tube of Quick Ligase Buffer, and use one small aliquot each time. 

ordering high quality HPLC purified adaptor will help reduce self-ligated adaptor sequences, thus eliminate the 125bp chimeric band
5x Kapa Ligation buffer         10ul
DNA ligase                  5ul
DNA adaptor (30uM)          5ul
A tailed DNA               30ul

Incubate at 20C for 20 minutes
purify the product with Ampure beads (1X) and elute in 42ul ddH2O

PCR amplification[edit]

Ligation products                 10ul  
10uM PCR_F                         1ul        
10uM PCR_R_index                   1ul (designed by Dinh: [Link]
2X Phusion HF MasterMix           25ul     
H2O                               13ul       
4 well for each sample
PCR program: 98 °C 30sec  -> 7 cycles of (98°C 10sec -> 60°C 20 sec -> 72°C 15sec) -> 72°C 3min ->15°C hold.
purify the products with Qiaquick columns and elute in 60ul total per sample
Perform another bead purification (1x) for size selection purpose and elute in 42ul ddH2O total for each sample

Qubit dsDNA HS Assay[edit]

# Sample Sample Concentration (ng/ul)
1 bc1_parent_N2-1_seq_lib 16.2
2 bc1_control_n2-2_seq_lib 21
3 WGL-talen_C3_N2-3_seq_lib 21
4 WGL-Talen_C6_N2-4_seq_lib 19.5
5 WGL-hcas9_C4_N2-5_seq_lib 21.2
6 WGL-hcas9_C16_N2-6_seq_lib 17.7

PAGE Gel Quantification[edit]

File:ZhangLab 2 2014-01-16 12hr 50min.jpg

pooling the libraries for PAGE gel purification[edit]

Sample ID Sample Concentration (ng/ul) amount needed (ul)
WGL_bc1_parent_N2-1_seq_lib 16.2 10
WGL_bc1_control_n2-2_seq_lib 21 8
WGL-talen_C3_N2-3_seq_lib 21 8
WGL-Talen_C6_N2-4_seq_lib 19.5 8
WGL-hcas9_C4_N2-5_seq_lib 21.2 8
WGL-hcas9_C16_N2-6_seq_lib 17.7 9

PAGE size selection and purification[edit]

  • total amount from above is 51 ul, then add 9 ul H2O and 20 ul 6x loading dye to load onto 2 PAGE gel cassettes and 4 lanes total

File:ZhangLab 2 2014-01-16 16hr 26min-edit.jpg

size select DNA smear in the range of 300 bp to 700 bp, shear the gel pieces through 0.5 ml tube(pierced with G20 gauge needle)
add 450 ul of 1x TE buffer
shake in 37C for 1 hour
centrifuge the tube and transfer the supernatant with limited amount of gel pieces onto Nanosap columns
recover ~420 ul of product and purify with ethanol precipitation

Quantification and calculation for sequencing[edit]

  • PAGE gel validation:
File:ZhangLab 2 2014-01-17 12hr 52min-edit.jpg