Library Prep for NextGen Sequencing

Isolation of genomic DNA from zebrafish larvae for sequencing libraries

Preparing Embryos or Larvae

1. Separate embryos or larvae (I like 100 d5) per 50mL conical tube
2. Remove all egg water
3. If larvae are hatched, continue with step 9. Else, to dechorionate, add ~500uL pronase to each sample)
4. Incubate 3 min. @ RT (gently shake at beginning of incubation

for 3-4 sec.)

1. Add Ca-free Ringer's up to the 50mL mark, squirting gently along

side of tube using plastic squeeze bottle. Add a little at a time to each tube instead of adding total volume to each tube at once.

1. Pour off most of Ringer's, repeat wash, being gentle and

squirting along sides of tubes

1. Repeat wash and pour off Ringer's for a total of three washes.
2. Remove as much supernatant as possible and gently transfer to

1.5mL tubes with transfer pipette

1. Wash once with 1X PBS, then remove as much as possible
2. Add 50 ul per larva, up to 1 ml DNA extraction buffer and

homogenize with a 3 ml syringe (20-23G needle), at least 10 passes.

DNA Extraction buffer (makes 10 ml)

final conc.	stock	volume
10 mM Tris pH 8.2	1M	100 ul
10 mM EDTA	0.5M	200 ul
200 mM NaCl	1M	2 ml
0.5% SDS	10 %	500 ul
200 µg/ml proteinase K	20 mg/ml	100 ul
H2O		7.1 ml

1. Incubate @37C for 1-2 hrs (until fully dissolved), vortexing briefly every 30 min
2. Add 0.6 ml isopropanol per tube and incubate @ -20C for at least

30 min (I like >2 hrs).

1. Centrifuge @4C for 15 min, max speed.
2. Wash with 1 ml of 70% EtOH, then centrifuge again @4C for 5 min,

max speed.

1. Remove EtOH by pipetting, spin briefly, remove remaining EtOH.
2. Resuspend in 100 ul TE. Keep on ice. Freeze or proceed to

shearing.

DNA fragmentation using the Covaris

1. Shear for 200nt size using the covaris (program: 200nt_nextGen

(3 min), Biopolymers Facility).

1. Transfer sample from covaris tube into a 1.5 ml low-retention

Eppi, add 1 ul of 20 mg/ ml RNase A ("Purelink", Invitrogen).

1. Incubate for 1 hr @ 37C.

MinElute PCR Reaction Cleanup kit

1. transfer sample into 1.5 ml low-bind eppendorf tube
2. add 500 ul of buffer PB to sample
3. Place MinElute column in and add sample to column
4. Wait 2 minutes, spin @ 13,000 rpm, discard the flow-through
5. Add 750 ul of buffer PE to column
6. Wait 1 minutes, spin @ 13,000 rpm for 2 min, discard the

flow-through

1. Spin again @ 13,000 rpm for 2 min, discard the flow-through
2. place column in clean 1.5 ml Eppi
3. Open column and air dry for 2 min
4. Add 15 ul of buffer EB to column, let sit for 2 min, spin for 60

sec

1. Add 15 ul of buffer EB to column, let sit for 2 min, spin for 60

sec

Perform End Repair

1. Prepare reaction mix:

30 ul	DNA sample
45 ul	nuclease-free water
10 ul	T4 DNA Ligase buffer w/ 10mM ATP (Phosphorylation Reaction Buffer)
4 ul	dNTP Mix
5 ul	T4 DNA Polymerase
1 ul	Klenow DNA Polymerase, Large Fragment
5 ul	PNK T4
100 ul	final vol.

1. Incubate in thermal cycler for 30 min at 20C

MinElute PCR Reaction Cleanup kit

1. transfer sample into 1.5 ml low-bind eppendorf tube
2. add 500 ul of buffer PB to sample
3. Place MinElute column in and add sample to column
4. Wait 1 minutes, spin @ 13,000 rpm, discard the flow-through
5. Add 750 ul of buffer PE to column
6. Wait 1 minutes, spin @ 13,000 rpm for 1 min, discard the

flow-through

1. Spin again @ 13,000 rpm for 1 min, discard the flow-through
2. place column in clean 1.5 ml Eppi
3. Add 32 ul of buffer EB to column, let sit for 1 min, spin for 1

min @ 13,000 rpm

Add "A" Bases to Fragments

1. Prepare reaction mix:

32 ul	DNA sample
5 ul	Klenow Buffer (NEBuffer 2)
10 ul	1 mM ATP
3 ul	Klenow Fragment, Exo-

1. Incubate 30 minutes @ 37C

MinElute PCR Reaction Cleanup kit

1. transfer sample into 1.5 ml low-bind eppendorf tube
2. add 250 ul of buffer PB to sample
3. Place MinElute column in and add sample to column
4. Wait 1 minutes, spin @ 13,000 rpm, discard the flow-through
5. Add 750 ul of buffer PE to column
6. Wait 1 minutes, spin @ 13,000 rpm for 1 min, discard the

flow-through

1. Spin again @ 13,000 rpm for 1 min, discard the flow-through
2. place column in clean 1.5 ml Eppi
3. Add 10 ul of buffer EB to column, let sit for 1 min, spin for 1

min @ 13,000 rpm

Ligate Adaptors to Fragments and Purify

1. Prepare reaction mix:

10 ul	DNA sample
25 ul	2x DNA Ligase Buffer (Quick Ligation Reaction Buffer)
10 ul	Adapter Oligo (maintain a 10:1 adapter/insert ratio - 5ug/10ul)
5 ul	DNA Ligase (Quick T4 DNA Ligase)

1. Incubate at RT for 15 minutes

MinElute PCR Reaction Cleanup kit

1. transfer sample into 1.5 ml low-bind eppendorf tube
2. add 250 ul of buffer PB to sample
3. Place MinElute column in and add sample to column
4. Wait 1 minutes, spin @ 13,000 rpm, discard the flow-through
5. Add 750 ul of buffer PE to column
6. Wait 1 minutes, spin @ 13,000 rpm for 1 min, discard the

flow-through

1. Spin again @ 13,000 rpm for 1 min, discard the flow-through
2. place column in clean 1.5 ml Eppi
3. Add 32 ul of buffer EB to column, let sit for 1 min, spin for 1

min @ 13,000 rpm

Size Selection on 1-1.5% Agarose Gel and QuiaQuick Gel extraction

1. Prepare 1-1.5% Agarose gel
2. Load Sample and run for 30 min @ >150V
3. Cut out band (~266 bp) and weigh gel slice
4. Add 3 gel volumes of Buffer QG and melt @50C for 10 min
5. Add 1 gel volume of isopropanol (add 1-2 ul of 3M NaAc, pH5.5 if

buffer is orange to purple)

1. Place MinElute column in and add sample to column
2. Wait 2 minutes, spin @ 13,000 rpm, discard the flow-through
3. Add 750 ul of buffer PE to column
4. Wait 1 minutes, spin @ 13,000 rpm for 1 min, discard the

flow-through

1. Spin again @ 13,000 rpm for 1 min, discard the flow-through
2. place column in clean 1.5 ml Eppi
3. Add 10 ul of buffer EB to column, let sit for 1 min, spin for 1

min @ 13,000 rpm

Enrich the Adaptor-Modified DNA Fragments by PCR

1. Prepare reaction mix:

1 ul	DNA sample
25 ul	2x Flash Phusion Master Mix (Phusion High Fidelity PCR Master Mix, Finnzymes)
1 ul	PCR primer 1.1
1 ul	PCR primer 2.1
22 ul	Nuclease-free water

1. Run the following PCR program:

Cycles	Duration	Temperature
1	30 sec	98C
10
-	10 sec	98C
-	30 sec	65C
-	30 sec	72C
1	5 min	72C
1	hold	4C

Size Selection on 1-1.5% Agarose Gel and QuiaQuick Gel extraction (optional: can be purified on E-gel by Biopolymers Facility)

1. Prepare 1-1.5% Agarose gel
2. Load Sample and run for 30 min @ >150V
3. Cut out band (~266 bp) and weigh gel slice
4. Add 3 gel volumes of Buffer QG and melt @50C for 10 min
5. Add 1 gel volume of isopropanol (add 1-2 ul of 3M NaAc, pH5.5 if

buffer is orange to purple)

1. Place MinElute column in and add sample to column
2. Wait 2 minutes, spin @ 13,000 rpm, discard the flow-through
3. Add 750 ul of buffer PE to column
4. Wait 1 minutes, spin @ 13,000 rpm for 1 min, discard the

flow-through

1. Spin again @ 13,000 rpm for 1 min, discard the flow-through
2. place column in clean 1.5 ml Eppi
3. Add 30 ul of buffer EB to column, let sit for 1 min, spin for 1

min @ 13,000 rpm

1. Submit library for quality control and sequencing