TITLE: Tn5 based tagmentation library prep protocol, high throughput 
AUTHORS: Charlotte Grace Sprehn, Erik Enbody, Yanjun Zan, Leif Andersson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>A very cost effective library preparation for whole-genome Illumina-based sequencing was previously demonstrated by by Picelli et al (Picelli et al., 2014). In this protocol, we optimized the Picelli et al protocol for very high throughput and is capable of generating libraries in 2x 96 well plates every 2 days. This protocol is adapted slightly from a protocol  by Zan and Carlborg for high throughput library preparation (</span><a href="https://dx.doi.org/10.17504/protocols.io.rt8d6rw" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://dx.doi.org/10.17504/protocols.io.rt8d6rw</span></a><span>), but optimized for a project focused on high throughput of Darwin's finch samples (</span><a href="https://www.biorxiv.org/content/10.1101/2021.01.19.426595v1" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.biorxiv.org/content/10.1101/2021.01.19.426595v1</span></a><span>). </span></div></div>

[STEPS]
?. [Transposon Assembly]
Remove primers Tn5ME-A, Tn5ME-B, Tn5ME-Arev, Tn5ME-Brev, DF buffer, and Tn5 from freezer. Melt primers and buffer, mix by vortexing and spin. Leave Tn5 to melt on bench. Turn on  incubator.
70 °C
?. [Transposon Assembly]
Prepare primers A and B in  tube: primer Tn5ME-A +  primer Tn5ME-rev = primer A () primer Tn5ME-B +  primer Tn5ME-rev = primer B ()
1.5 mL
27.5 µl
27.5 µl
55 µl
27.5 µl
27.5 µl
55 µl
?. [Transposon Assembly]
Incubate at  for  then place .
70 °C
on ice
?. [Transposon Assembly]
In a new  tube combine the following: Tn5 () primer A primer B 2x DF bufferTotal	Mix by pipetting.
1.5 mL
85 µl
55 µl
55 µl
110 µl
305 µl
?. [Transposon Assembly]
Incubate at  for .
0 Room temperature
?. [Tagmentation]
Start thermocyclers so they are up to temp. Program: ,  for  volume.
55 °C
55 °C
15 µl
?. [Tagmentation]
In new  tube add:                        (1x)900uL H2O4.5uL400uL 5x TAPS buffer2uL400uL 40% PEG2uL200uL Tn5 mix1uLDNA (10ng/uL)1uLTotal 1900uL mixMix by inverting. Aliquot  into a strip of 8, and use strip to aliquot  into 2x 96-well plates using multichannel. If DNA is of questionable quality the volume can be increased to 1.5 or more, just scale water accordingly.
2 mL
900uL H2O4.5uL400uL 5x TAPS buffer2uL400uL 40% PEG2uL200uL Tn5 mix1uLDNA (10ng/uL)1uLTotal 1900uL mix
225 µl
9 µl
?. [Tagmentation]
Move to DNA bench. Spin thawed DNA dilution plates. Add  DNA to prepared plates of mix. Seal with film and spin.
1 µl
?. [Tagmentation]
Incubate in thermocycler  at .
55 °C
?. [Tagmentation]
Remove from thermocycler. Carefully remove film and add  0.2% SDS (prepared in a strip tube). Seal with new film and return to thermocyclers to incubate for .
2.5 µl
?. [PCR Enrichment]
Remove KAPA HiFi dNTPs and GC buffer from freezer and thaw, vortex, and spin. Index plates should be thawed and spun down.
KAPA HiFi PCR enzyme should only be out of the freezer briefly.
?. [PCR Enrichment]
In a  tube mix the following:                                      (1x)576uL H2O3uL&nbsp;960uL 5x PCR buffer5uL57.6uL dNTPs (10mM)0.3uL38.4uL HiFi PCR enzyme0.2uL&nbsp;Total 1632uL Mix by inverting and spin.
2 mL
576uL H2O3uL&nbsp;960uL 5x PCR buffer5uL57.6uL dNTPs (10mM)0.3uL38.4uL HiFi PCR enzyme0.2uL&nbsp;Total 1632uL
?. [PCR Enrichment]
Aliquot  into an 8-strip. Move to DNA bench. Add  of mix to plates from tagmentation (which have  of product, so will end with ).
200 µl
7.5 µl
12.5 µl
20 µl
?. [PCR Enrichment]
Add index. Index plates are pre-prepared with equal amounts of both indices, so add  of the mix (=2.5uL each index) at  each.
5 µl
Be careful that plate positions match!
?. [PCR Enrichment]
Cover PCR plates with lids (not film), spin, and place in thermocyclers for the following program:
?. [PCR Enrichment]
When complete freeze at  or proceed to cleanup.
-20 °C
?. [Bead Cleanup]
Remove beads from cold room and let sit at  for . Mix by inversion, light shaking before use. Pour about  into boat.
0 Room temperature
2.6 mL
?. [Bead Cleanup]
Add  (with filter tips) to each well of PCR product. Cover with film and shake by hand until completely homogenous. Incubate at . Spin briefly.
7.5 µl
0 Room temperature
?. [Bead Cleanup]
Place on magnet stands for  until all beads are on the sides.
?. [Bead Cleanup]
Pipette  to new plates. Pipette opposite the beads so as not to disturb them.
28 µl
?. [Bead Cleanup]
Add  beads (stir beads with pipette tip first). Cover with film, shake, and incubate  at . Spin briefly.
4.5 µl
0 Room temperature
?. [Bead Cleanup]
Place on magnet stand for .
?. [Bead Cleanup]
While still on magnet, pipette out and throw away  of liquid-don’t touch beads. About  remains.
22.5 µl
10 µl
?. [Bead Cleanup]
Make fresh 80% EtOH and add  to each. Place large stack of paper towels over the top and invert (towels, plate, and magnet) and shake. Leave upside down on towels for about a .
70 µl
?. [Bead Cleanup]
Repeat #24, but before dumping lift plate from magnet and allow all beads to drop to the bottoms of the tubes. Place back on magnets and leave for  before dumping. Shake vigorously and leave plate and magnet on its side on the bench to dry for about . Let EtOH evaporate but do not leave too long, ensure no cracking in bead dot.
?. [Bead Cleanup]
Add  sterile H2O to each.
22 µl
?. [Bead Cleanup]
Cover with film, remove from magnets and shake by hand on trays vigorously. Spin briefly and put on shaker  @ 2000. Incubate at . Spin.
0 Room temperature
?. [Bead Cleanup]
Place on magnets for . Prepare new plates during incubation.
?. [Bead Cleanup]
Remove film and pipette  into new plates. Seal with lids. Proceed to QC or place in cold room.
19 µl
?. [QC & Pool]
Use a fluorescence plate reader to quantify the amount of DNA in the two plates.
We used a Tecan (Switzerland) plate reader and calibrated using a Qubit.
?. [QC & Pool]
Pool accordingly for equimolar libraries. Store pools in cold room or freeze at  if not doing final wash within a day.
-20 °C
Pool according to the required concentration needed for the sequencing platform. We usually made dilution plates to ~1.5ng/uL for Illumina NovaSeq applications, but changed slightly depending on the lowest concentration samples in the plates. Plan to lose ~half measurement from post-pooling wash.
?. [Post-pooling wash]
Take beads out to bring to  before starting. Mix thoroughly.
0 Room temperature
?. [Post-pooling wash]
Calculate how much of each pool you need to start with   or as needed for your sequencing. Calculate how much beads you need for each pool (.45x and .3x).
1000 ng
Adjust for how much you need to send for sequencing. You’ll end up with less than half of this starting concentration.
?. [Post-pooling wash]
Transfer calculated amount of each pool into  tubes.
2 mL
?. [Post-pooling wash]
Add 0.45x AmpPure beads. Mix by gentle pipetting until homogenous.
?. [Post-pooling wash]
Incubate . Put remaining pools back in cold room.
0 Room temperature
?. [Post-pooling wash]
Place tubes on tube magnet block for . Label new  tubes.
2 mL
?. [Post-pooling wash]
Transfer supernatant to new tubes. Pipette away from beads.
?. [Post-pooling wash]
Add 0.3x beads. Mix until homogenous.
?. [Post-pooling wash]
Incubate  at .
0 Room temperature
?. [Post-pooling wash]
Place on magnet .
?. [Post-pooling wash]
Pipette off supernatant. Leave tubes open on magnet.
?. [Post-pooling wash]
Wash beads with  fresh 80% EtOH. Leave a couple seconds and pipette off EtOH into waste.
500 µl
?. [Post-pooling wash]
Let dry  at . Aliquot some sterile H2O into a  tube.
0 Room temperature
2 mL
?. [Post-pooling wash]
Suspend beads in  H2O, vortex well.
100 µl
?. [Post-pooling wash]
Place on magnet .
?. [Post-pooling wash]
Move supernatant to new tubes.
?. [Post-pooling wash]
Measure product on Qubit and Tapestation as desired. Adjust volumes to needed concentration via speedvac if needed. If too low can repeat this section with higher starting amount.