TITLE: Modified LSK109 ligation prep with needle shear and bead clean up
AUTHORS: John Tyson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Modified LSK109 ligation prep using needle shear and bead purification</span></div><div class = "text-block">Using HMW DNA purified as above we have been performing 29G and 26G needle shears to produce fragmentation to around 18Kb and 34kb respectively (reported read N50’s after basecalling). Also by going into the library preparations with 5-10ug of DNA have been able to produce enough material to provide up to 10 library loads from a single prep. We have not been performing the FFPE repair during the end-prep step, and have been diluting the sample at the cleanup points in the protocol with EB to prevent bead clumping during 0.4-0.5x AMPureXP purifications when DNA fragments are large and at high concentrations. Shown below are the results from a batch of 12 runs we performed using this approach.</div><div style = "text-align :; float : ;"><img style = "" src = "https://protocols-files.s3.amazonaws.com/private/1618c1a37f153002e3885d7ebd72cd0a4e653895bbeb80a8934c28142d836cb2/bq947vcw.jpg?X-Amz-Content-Sha256=UNSIGNED-PAYLOAD&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIAJYFAX46LHRVQMGOA%2F20200129%2Fus-east-1%2Fs3%2Faws4_request&X-Amz-Date=20200129T214513Z&X-Amz-SignedHeaders=host&X-Amz-Expires=604800&X-Amz-Signature=dcc06a559b530c8fdf0c28dfe4e4afe1cfaf6e3efcdb8182080a2b9db541d3c8" /></div></div>

[STEPS]
?. of HMW genomic DNA made up to  with EB and sheared with either a 29G or 26G needle using 20 passes. For 26G shear add  of EB (10mM Tris-Cl pH8.0) followed by AMPureXP bead solution.  For 29G shear add  AMPureXP bead solution.
10 µg
250 µl
250 µl
250 µl
125 µl
After shearing is a good time to get a reliable DNA quantification using the Qubit DNA HS kit etc.
?. Mix by flicking.
?. Remove supernatant and keeping on the magnet wash 2x with  –  80 % ethanol without disturbing the pellet.
300 µl
500 µl
?. Removed tube from the magnet
?. Place tube on magnet and remove eluted DNA sample to a fresh tube.
?. Quantify  of the DNA sample using the Qubit DNA HS kit to confirm DNA recovery (~).
1 µl
6 µg
?. Incubate at  for  followed by  for .
20 °C
65 °C
?. Add  EB buffer followed by  AMPureXP bead solution.
120 µl
72 µl
?. Mix by flicking.
?. Remove supernatant and keeping on the magnet wash 2x with  80 % ethanol without disturbing the pellet.
300 µl
?. Remove tube from the magnet.
?. Place tube on magnet and remove eluted DNA sample to a fresh tube.
?. Quantify  DNA sample using the Qubit DNA HS kit to confirm DNA recovery (~ – ).
1 µl
4 µg
5 µg
?. Incubate at  /  for .
20 °C
0 Room temperature
?. Add  EB buffer followed by  AMPureXP bead solution.
100 µl
80 µl
?. Mix by flicking.
?. Remove supernatant and keeping on the magnet wash 2x with of LFB buffer by removing from the magnet and flicking, briefly spin, and pelleting again on the magnet.
250 µl
?. Remove tube from the magnet.
?. Place tube on magnet and pellet beads.
?. Quantify  DNA sample using the Qubit DNA HS kit to confirm DNA recovery (~).
1 µl
2.5 µg
?. Library ready to be combined with SQB and LLB for loading onto flowcel ( –  per load).
200 ng
400 ng
?. Incubate for  at  followed by pelleting on a magnet.
0 Room temperature
?. Briefly spin, return to magnet and remove any residual 80 % ethanol with a pipette before allowing the bead pellet to air dry for ~.
?. Resuspend the bead pellet in  EB by flicking.
51 µl
?. Spin down.
?. Allow DNA to elute by incubation at  for .
37 °C
?. Then set up the following End-Prep reaction: DNA sample Ultra II End-Prep Buffer  Ultra II End-Prep Enzyme mix
50 µl
7 µl
3 µl
?. Incubate for  at  followed by pelleting on a magnet.
0 Room temperature
?. Briefly spin.
?. Return to magnet and remove any residual 80 % ethanol with a pipette before allowing the bead pellet to air dry for ~.
?. Resuspend the bead pellet in  EB by flicking.
67 µl
?. Spin down and allow DNA to elute by incubation at  for  with occasional flicking.
0 °C
?. Then set up the following Ligation reaction: DNA sample LNB buffer AMX adapter Quick T4 DNA Ligase
66 µl
25 µl
5 µl
4 µl
?. Briefly spin.
?. Incubate for  at  followed by pelleting on a magnet.
0 Room temperature
?. Remove any residual LFB buffer while on the magnet with a pipette after final pelleting.
?. Resuspend the bead pellet in  ONT-EB buffer by flicking.
21 µl
?. Spin down and allow DNA to elute by incubation at  for .
37 °C
?. Remove eluted DNA sample to a fresh tube.