Editing Library Prep for NextGen Sequencing

= Isolation of genomic DNA from zebrafish larvae for sequencing libraries  =



== Preparing Embryos or Larvae  ==

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

#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. 
#Pour off most of Ringer's, repeat wash, being gentle and 
squirting along sides of tubes 

#Repeat wash and pour off Ringer's for a total of three washes. 
#Remove as much supernatant as possible and gently transfer to 
1.5mL tubes with transfer pipette 

#Wash once with 1X PBS, then remove as much as possible 
#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) 

{| cellspacing="1" cellpadding="1" border="0"
|-
| bgcolor="#eaeaea" | final conc. 
| bgcolor="#eaeaea" | stock 
| bgcolor="#eaeaea" | volume
|-
| bgcolor="#ffffff" | 10 mM Tris pH 8.2 
| bgcolor="#ffffff" | 1M 
| bgcolor="#ffffff" | 100 ul
|-
| bgcolor="#eaeaea" | 10 mM EDTA 
| bgcolor="#eaeaea" | 0.5M 
| bgcolor="#eaeaea" | 200 ul
|-
| bgcolor="#ffffff" | 200 mM NaCl 
| bgcolor="#ffffff" | 1M 
| bgcolor="#ffffff" | 2 ml
|-
| bgcolor="#eaeaea" | 0.5% SDS 
| bgcolor="#eaeaea" | 10&nbsp;% 
| bgcolor="#eaeaea" | 500 ul
|-
| bgcolor="#ffffff" | 200 µg/ml 
proteinase K 

| bgcolor="#ffffff" | 20 mg/ml 
| bgcolor="#ffffff" | 100 ul
|-
| bgcolor="#eaeaea" | H2O 
| bgcolor="#eaeaea" | &nbsp; 
| bgcolor="#eaeaea" | 7.1 ml
|}



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

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

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




== DNA fragmentation using the Covaris  ==



#Shear for 200nt size using the covaris (program: 200nt_nextGen 
(3 min), Biopolymers Facility). 

#Transfer sample from covaris tube into a 1.5 ml low-retention 
Eppi, add 1 ul of 20 mg/ ml RNase A ("Purelink", Invitrogen). 

#Incubate for 1 hr @ 37C.



== MinElute PCR Reaction Cleanup kit  ==



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

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

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




== Perform End Repair  ==



#Prepare reaction mix:

{| cellspacing="1" cellpadding="1" border="0"
|-
| bgcolor="#eaeaea" | 30 ul 
| bgcolor="#eaeaea" | DNA sample
|-
| bgcolor="#ffffff" | 45 ul 
| bgcolor="#ffffff" | nuclease-free water
|-
| bgcolor="#eaeaea" | 10 ul 
| bgcolor="#eaeaea" | T4 DNA Ligase buffer w/ 10mM ATP 
(Phosphorylation Reaction Buffer) 

|-
| bgcolor="#ffffff" | 4 ul 
| bgcolor="#ffffff" | dNTP Mix
|-
| bgcolor="#eaeaea" | 5 ul 
| bgcolor="#eaeaea" | T4 DNA Polymerase
|-
| bgcolor="#ffffff" | 1 ul 
| bgcolor="#ffffff" | Klenow DNA Polymerase, Large Fragment
|-
| bgcolor="#eaeaea" | 5 ul 
| bgcolor="#eaeaea" | PNK T4
|-
| bgcolor="#ffffff" | 100 ul 
| bgcolor="#ffffff" | final vol.
|}



#Incubate in thermal cycler for 30 min at 20C



== MinElute PCR Reaction Cleanup kit  ==



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

#Spin again @ 13,000 rpm for 1 min, discard the flow-through 
#place column in clean 1.5 ml Eppi 
#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  ==



#Prepare reaction mix:

{| cellspacing="1" cellpadding="1" border="0"
|-
| bgcolor="#eaeaea" | 32 ul 
| bgcolor="#eaeaea" | DNA sample
|-
| bgcolor="#ffffff" | 5 ul 
| bgcolor="#ffffff" | Klenow Buffer (NEBuffer 2)
|-
| bgcolor="#eaeaea" | 10 ul 
| bgcolor="#eaeaea" | 1 mM ATP
|-
| bgcolor="#ffffff" | 3 ul 
| bgcolor="#ffffff" | Klenow Fragment, Exo-
|}



#Incubate 30 minutes @ 37C



== MinElute PCR Reaction Cleanup kit  ==



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

#Spin again @ 13,000 rpm for 1 min, discard the flow-through 
#place column in clean 1.5 ml Eppi 
#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  ==



#Prepare reaction mix:

{| cellspacing="1" cellpadding="1" border="0"
|-
| bgcolor="#eaeaea" | 10 ul 
| bgcolor="#eaeaea" | DNA sample
|-
| bgcolor="#ffffff" | 25 ul 
| bgcolor="#ffffff" | 2x DNA Ligase Buffer (Quick Ligation 
Reaction Buffer) 

|-
| bgcolor="#eaeaea" | 10 ul 
| bgcolor="#eaeaea" | Adapter Oligo (maintain a 10:1 
adapter/insert ratio - 5ug/10ul) 

|-
| bgcolor="#ffffff" | 5 ul 
| bgcolor="#ffffff" | DNA Ligase (Quick T4 DNA Ligase)
|}



#Incubate at RT for 15 minutes



== MinElute PCR Reaction Cleanup kit  ==



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

#Spin again @ 13,000 rpm for 1 min, discard the flow-through 
#place column in clean 1.5 ml Eppi 
#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  ==



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

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

#Spin again @ 13,000 rpm for 1 min, discard the flow-through 
#place column in clean 1.5 ml Eppi 
#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  ==



#Prepare reaction mix:

{| cellspacing="1" cellpadding="1" border="0"
|-
| bgcolor="#eaeaea" | 1 ul 
| bgcolor="#eaeaea" | DNA sample
|-
| bgcolor="#ffffff" | 25 ul 
| bgcolor="#ffffff" | 2x Flash Phusion Master Mix (Phusion High 
Fidelity PCR Master Mix, Finnzymes) 

|-
| bgcolor="#eaeaea" | 1 ul 
| bgcolor="#eaeaea" | PCR primer 1.1
|-
| bgcolor="#ffffff" | 1 ul 
| bgcolor="#ffffff" | PCR primer 2.1
|-
| bgcolor="#eaeaea" | 22 ul 
| bgcolor="#eaeaea" | Nuclease-free water
|}



#Run the following PCR program:



{| cellspacing="1" cellpadding="1" border="0"
|-
| bgcolor="#eaeaea" | Cycles 
| bgcolor="#eaeaea" | Duration 
| bgcolor="#eaeaea" | Temperature
|-
| bgcolor="#ffffff" | 1 
| bgcolor="#ffffff" | 30 sec 
| bgcolor="#ffffff" | 98C
|-
| bgcolor="#eaeaea" | 10 
| bgcolor="#eaeaea" | &nbsp; 
| bgcolor="#eaeaea" | &nbsp;
|-
| bgcolor="#ffffff" | - 
| bgcolor="#ffffff" | 10 sec 
| bgcolor="#ffffff" | 98C
|-
| bgcolor="#eaeaea" | - 
| bgcolor="#eaeaea" | 30 sec 
| bgcolor="#eaeaea" | 65C
|-
| bgcolor="#ffffff" | - 
| bgcolor="#ffffff" | 30 sec 
| bgcolor="#ffffff" | 72C
|-
| bgcolor="#eaeaea" | 1 
| bgcolor="#eaeaea" | 5 min 
| bgcolor="#eaeaea" | 72C
|-
| bgcolor="#ffffff" | 1 
| bgcolor="#ffffff" | hold 
| bgcolor="#ffffff" | 4C
|}



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



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

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

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

#Submit library for quality control and sequencing

[[Category:Protocols]]