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]
File:ZhangLab 2 2014-01-17 12hr 52min-edit.jpg