TITLE: InDrop Library Preparation - TruDrop (modified V2) Detailed
AUTHORS: Austin Southard-Smith, Alan Simmons, Ken Lau

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details the library preparation process of dual-indexed, NovaSeq-compatible, single-cell RNA-Seq libraries from the inDrop V2 platform. The final library structure combines the TruSeq sequencing primers and indices from standard illumina libraries with the inDrop V2 library features such as the cell barcodes, unique molecular identifier, and transcript. As such it is referred to TruSeq-inDrop (TruDrop).</div></div>

[STEPS]
?. [Oligo Removal]
If necessary, thaw samples on ice.
?. [Oligo Removal]
While samples are thawing, visually estimate the volume of each sample. Record this value
?. [Oligo Removal]
For every 70 uL of post-RT material prepare 100 uL of Digest mix as follows:Component:Ultrapure RNase/DNase free H2O                                10X Fast Digest Buffer                                    Exonuclease I
86 µl
9 µl
5 µl
While the samples are thawing mix the H2O and digest buffer. Then add and mix the Exonuclease I to the Digest mix while the sample are spinning in step 4.
?. [Oligo Removal]
Centrifuge tubes at   for   at  to pellet residual HFE (oil).
4 °C
Centrifuge: 16000 34
?. [Oligo Removal]
Adjust the pipette to the estimated sample volume and then block pipette tip with digest mix prior to using it to transfer the sample.
?. [Oligo Removal]
Without drawing up HFE-7500 oil from the bottom of the tube, carefully transfer the sample to a Costar Spin-X column.
Once the sample has been drawn into the pipette tip carefully adjust the pipette volume to the exact sample volume. Record this volume. Then eject the sample onto the column. Eject the sample directly above the filter column. Do not let the sample run down the sides of the filter column.
?. [Oligo Removal]
This marks the start of Day 1:Before starting do the following:Cool bench-top microcentrifuge to .Adjust a heat block to .
4 °C
37 °C
?. [Oligo Removal]
Add  of digest mix to the filter column for every  of sample. Recommended to record the volume added.
100 µl
70 µl
Do not stick the pipette tip into the sample volume. Pipette the digest mix out directly above the sample.
?. [Oligo Removal]
Repeat steps 7 — 8 for all samples.
?. [Oligo Removal]
Add the appropriate volume of AMPure Beads to each sample and mix by pipetting until no mixing lines are observed (use the same tip that was used to add the AMPure beads to mix the sample). DO NOT VORTEX THE SAMPLE.
?. [Oligo Removal]
For each sample pre-chill 1  DNA LoBind Tube and 1 Spin-X Column + Tube . Move a 2 mL aliquot of AMPure XP beads to  to start equilibrating. If a heat
1.5 mL
on ice
0 Room temperature
?. [Oligo Removal]
Centrifuge filter column and collection tube at   for   at  to pass sample through the filter column.
4 °C
Centrifuge: 16000 34
?. [Oligo Removal]
Place the samples back . Adjust the pipet tip to the total volume. Block tip with digest mix. Carefully transfer the sample from the collection tube to 1.5 mL DNA LoBind tube.
on ice
Once the sample has been drawn into the pipette tip carefully adjust the pipette volume to the exact sample volume. Record this volume. Then eject the sample into the 1.5mL DNA LoBind tube. Eject the sample at the bottom of the tube. Do not let the sample run down the sides of the tube.
?. [Oligo Removal]
Incubate samples at  for .
37 °C
During this incubation place the 10X Second Strand Synthesis Reaction Buffer (10X SSS buffer)   to start thawing.
?. [Oligo Removal]
Vortex the AMPure bead aliquot to resuspend AMPure beads (5-10 seconds is sufficient).
?. [Oligo Removal]
During this spin use the previously measured sample volume and digest mix volume to calculate the total volume
?. [Oligo Removal]
During the incubation calculate the volume of AMPure XP Beads that will be used in the following 0.8x AMPure purification (AMPurification).
Example: (  sample volume) x 0.8 =   AMPure XP Beads
?. [Oligo Removal]
Incubate each sample/bead mixture at  for .
0 Room temperature
?. [Oligo Removal]
Place each sample tube on a magnetic tube rack for  at  or until the beads have fully separated from the supernatant.
0 Room temperature
?. [Oligo Removal]
Carefully aspirate the supernatant without removing/disturbing the bead pellet.
While doing this, slowly pipette from the side of each tube that is opposite the bead pellet. Do not touch the beads with the pipette tip.
?. [Oligo Removal]
Wash the Bead pellet with  ethanol twice at .
0 Room temperature
?. [Oligo Removal]
After the second ethanol wash quick-spin the sample tubes in a  mini-centrifuge for   at  to collect any residual ethanol at the bottom of the tube.
0 Room temperature
?. [Oligo Removal]
Place the tubes back on the magnet rack and then use a P200 or smaller pipette to remove any residual ethanol from the bottom of the tube. Do not touch the bead pellet!
?. [Oligo Removal]
For a single sample: Keeping the tube on the magnet rack gently wash the beads with  of  ethanol. Rotate each tube 180°, allowing the beads to re-pellet on the side now closest to the magnet.
400 µl
Pipette the ethanol onto the opposite side of the tube from the beads so as to not greatly disturb the bead pellet. If some beads do not passing from one side of the tube to the other this can sometimes be remedied by taping on the side of the tube where the beads are stuck. If this does not work it is recommended to use the beads that do pass to the other side of the tube to loosen the beads that are stuck by slowly moving the tube around on the magnet such that the beads that were stuck are closest to the magnet.
?. [Oligo Removal]
Repeat Step 19.1 for each sample.
?. [Oligo Removal]
Again rotate each tube 180°, allowing the beads to re-pellet on the side now closest to the magnet. All tubes should now be in their original orientation.
?. [Oligo Removal]
Use a pipette to aspirate and discard the Ethanol. Pipette slowly on the opposite side of the tube from the bead pellet. Again do not disturb the bead pellet with the pipette tip.
?. [Oligo Removal]
Repeat steps 19.1 - 19.4 to for the second  ethanol wash.
?. [Oligo Removal]
Leaving the tubes open on the magnetic tube rack allow each bead pellet to air dry (normally 5-10 min) at . While samples are drying get out 1 PCR tube for each sample (do not use strip tubes or plates) and pre-chill on the 96 well aluminum block .
0 Room temperature
on ice
Place a cover over the tubes to prevent dust from falling in them. The pellet will need to dry for at least 5 min. The bead pellet will be a matte black when it is dry and potentially starting to show 1 or 2 cracks. Do not allow the bead pellet to become completely cracked as this will reduce elution efficiency. It is completely normal for some sample to take longer to dry than others (usually larger samples take longer to dry).
?. [Oligo Removal]
Once a sample is dry add  Ultrapure DNase/RNase free H2O to just that dry sample and place on a normal tube rack at .
17 µl
0 Room temperature
?. [Oligo Removal]
Gently flick the bottom of the tube to re-suspend the bead pellet in the H2O.
Avoid flinging the solution onto the top half of the tube. Avoid flinging the solution onto the inside of the lid. DO NOT VORTEX!
?. [Oligo Removal]
Incubate the sample at  for  on a normal tube rack. Proceed to step 26 while waiting.
0 Room temperature
RNA/DNA will not elute if samples are kept cold during this step.
?. [Oligo Removal]
While the samples are eluting, mix the thawed 10X SSS buffer by pipetting and add  of 10X SSS buffer to each chilled PCR tube.
2 µl
?. [Oligo Removal]
Spin the sample tubes in a  mini-centrifuge for   at  to collect everything at the bottom of the tube.
0 Room temperature
?. [Oligo Removal]
Place the tube on the magnet rack for about   to pellet the beads on the side of the tube.
If not all of the beads pellet against the magnet that is okay. This is caused by either high protein content/debris left over from the encapsulation or a small amount of residual ethanol. The beads can be removed in the following step.
?. [Second Strand Synthesis (SSS)]
To each chilled PCR tube add 1 uL of NEBNext Second Strand Synthesis Enzyme mix. Mix the sample by pipetting until few to no mixing lines are observed (10 pipette up and down motions while stirring with the pipet).
?. [Oligo Removal]
Use a P20 pipette to transfer the supernatant of each sample to its respective chilled PCR tubes and mix by pipetting. If the sample volume is less than 17 uL adjust the volume of the sample up to 17 uL using a P2.
Carrying the magnetic AMPure beads forward should be avoided. Residual AMPure beads can be removed by pipetting the sample out onto the side of the 1.5mL DNA LoBind tube directly above the magnet. Slowly pipette the solution up and down 2-3 times to dislodge any beads stuck to the pipette tip. Allow the beads to pellet against the top of the magnet. Then slowly draw the supernatant back into that same pipette tip while being careful to avoid drawing up any beads that may be present. Throughout this entire process USE A SINGLE PIPETTE TIP PER SAMPLE.It is very important that the total volume added to the tube is 17 uL of sample in H2O going into SSS.
?. [Second Strand Synthesis (SSS)]
Start a PCR machine following the below protocol:  for   for  holdLid: Do not place the samples on the PCR machine until the PCR block is at . Once the PCR block temperature is at  the samples should be placed on the incubator to start the SSS reaction.
16 °C
65 °C
4 °C
50 °C
16 °C
16 °C
Proceed to preparing the In Vitro Transcription reaction while the SSS reaction is on-going.
?. [In Vitro Transcription (IVT)]
When the SSS reaction has 50 minutes remaining move the contents of the NEB HiScribe T7 High Yield RNA Synthesis Kit to an ice bucket to start thawing   (leave the T7 enzyme at ).
on ice
-20 °C
?. [In Vitro Transcription (IVT)]
When the SSS reaction has 10-15 minutes remaining prepare enough of the following IVT master mix   for each sample using the NEB HiScribe T7 Kigh Yield RNA Synthesis Kit reagents (mix by pipetting):        Reagent                                                    Volume/ReactionUltraPure DNase/Rnase-free H2O                        NEB T7 High Yield 10x rxn buffer                            ATP                                                                               CTP                                                                               GTP                                                                               UTP                                                                               NEB T7 High Yield Enzyme Mix                               Pyrophosphatase (NEB M0361S)
on ice
10 µl
8 µl
8 µl
8 µl
8 µl
8 µl
8 µl
2 µl
?. [In Vitro Transcription (IVT)]
When the SSS reaction has completed, quick-spin the samples in PCR tubes for  at .
0 Room temperature
?. [In Vitro Transcription (IVT)]
At  add  of IVT master mix to each sample's PCR tube. Mix by pipetting until no mixing lines are observed.
0 Room temperature
60 µl
?. [In Vitro Transcription (IVT)]
Start a PCR machine to the follow the below protocol: for Lid: Do not place the samples on the PCR machine until the PCR block is at . Once the PCR block temperature is at  the samples should be placed on the incubator to start the SSS reaction.
37 °C
50 °C
37 °C
37 °C
?. [In Vitro Transcription (IVT)]
Vortex the AMPure bead aliquot to resuspend the AMPure XP beads (5-10 seconds is sufficient for a   aliquot).
2 mL
?. [In Vitro Transcription (IVT)]
Add the 72 uL of AMPure Beads to each sample and mix by pipetting until no mixing lines are observed (use the same tip that was used to add the AMPure beads to mix the sample). DO NOT VORTEX THE SAMPLE.
?. [In Vitro Transcription (IVT)]
Incubate each sample/bead mixture at  for .
0 Room temperature
?. [In Vitro Transcription (IVT)]
Place each sample tube on a magnetic tube rack for  at  or until the beads have fully separated from the supernatant.
0 Room temperature
?. [In Vitro Transcription (IVT)]
Carefully aspirate the supernatant without removing/disturbing the bead pellet.
While doing this, slowly pipette from the side of each tube that is opposite the bead pellet. Do not touch the beads with the pipette tip.
?. [In Vitro Transcription (IVT)]
Wash the Bead pellet with  ethanol twice at .
0 Room temperature
?. [In Vitro Transcription (IVT)]
For a single sample: Keeping the tube on the magnet rack gently wash the beads with  of  ethanol. Rotate each tube 180°, allowing the beads to re-pellet on the side now closest to the magnet.
400 µl
Pipette the ethanol onto the opposite side of the tube from the beads so as to not greatly disturb the bead pellet. If some beads do not passing from one side of the tube to the other this can sometimes be remedied by taping on the side of the tube where the beads are stuck. If this does not work it is recommended to use the beads that do pass to the other side of the tube to loosen the beads that are stuck by slowly moving the tube around on the magnet such that the beads that were stuck are closest to the magnet.During the first Ethanol wash of the AMPurification after IVT it is normal to see a small cloud of white material precipitate out. This can be observed when drawing up and discarding the ethanol in subsequent steps.
?. [In Vitro Transcription (IVT)]
Repeat Step 43.1 for each sample.
?. [In Vitro Transcription (IVT)]
Again rotate each tube 180°, allowing the beads to re-pellet on the side now closest to the magnet. All tubes should now be in their original orientation.
?. [In Vitro Transcription (IVT)]
Use a pipette to slowly aspirate and discard the Ethanol. Draw the ethanol up from the opposite side of the bead pellet. Do not disturb the bead pellet with the pipette tip.
?. [In Vitro Transcription (IVT)]
Repeat steps 19.1 - 19.4 to for the second  ethanol wash.
?. [In Vitro Transcription (IVT)]
After the second ethanol wash, quick-spin the sample tubes in a  mini-centrifuge for   at  to collect any residual ethanol at the bottom of the tube.
0 Room temperature
?. [In Vitro Transcription (IVT)]
Place the tubes back on the magnet rack and then use a P200 or smaller pipette to remove any residual ethanol from the bottom of the tube. Do not touch the bead pellet!
?. [In Vitro Transcription (IVT)]
This marks the start of Day 2:Prepare an ice bucket (pack the ice).  chill the 96 well aluminum block. Prepare a single 1.5 mL DNA LoBind tube for each sample.
on ice
?. [In Vitro Transcription (IVT)]
Use 2 uL from the backup tube to measure the RNA concentration of the sample via Qubit using the Qubit RNA HS assay kit. At this point it is also optional to use 1 uL of the sample for evaluation using a BioAnalyzer RNA Pico Chip Assay.
The Qubit RNA HS reagent from the Qubit RNA HS assay kit is very light sensitive. Only use it with the lights off.
RNA yields are highly dependent on the types of cells and cell viability in the sample during encapsulation.For mouse/human intestinal epithelium cells:0-20 ng/uL RNA is low.20-60 ng/uL of RNA is normal.60+ ng/uL of RNA is high.If the RNA yield exceeds 85 ng/uL then the sample should be diluted to 80 ng/uL before proceeding to fragmentation.If the Qubit output is "TL" (i.e. Too Low) a library can still sometimes be prepared, but all of the sample (  )should be carried forwards ( ).
If samples will be further processed another day this is a safe pause point. Samples can be stored at  .
?. [Fragmentation]
According to 1CellBio: if the samples were run on a BioAnalyzer and the average size If samples were thawed from backup tubes transfer  to a PCR tube. Top off any missing volume with RNA Elution Buffer. Otherwise proceed to step 55.
9 µl
?. [Fragmentation]
Set up a thermal cycler to follow the below protocol:          holdlid:
70 °C
105 °C
?. [Fragmentation]
Prepare the following Fragmentation Stop mix in a DNA LoBind tube and keep at room temperature:   AB1ComponentVolume per Reaction2Ultrapure DNase/RNase-free H2O9 uL310x Fragmentation Stop Solution1 uL4AMPure Beads16 uL5Total26 uL
AB1ComponentVolume per Reaction2Ultrapure DNase/RNase-free H2O9 uL310x Fragmentation Stop Solution1 uL4AMPure Beads16 uL5Total26 uL
?. [Fragmentation]
For each sample Pre-chill a single 1.5mL DNA LoBind tube . Also pre-set a P200 to 26 uL. Steps 58-61 should be completed as quickly as possible after this.
on ice
?. [Fragmentation]
Warning: Wait to do this step until the thermal cycler is at . Add 1 uL of 10X RNA fragmentation reagent to each sample. Mix by pipetting (5 times) and stirring. Immediately place back on chilled 96 well aluminum block.
70 °C
?. [Fragmentation]
Once the fragmentation reagent has been added to all samples, place all samples on the pre-heated thermal cycler for 1-2 minutes. Refer to the below table for guidance with regards to fragmentation duration.  AB1IVT yield ranges (ng/uL)Example fragmentation durations for intestinal epithelium cell libraries (m:ss)20-20:00 to 0:2032-100:20 to 1:00410-401:00 to 1:30540-601:30 to 1:45660-801:40 to 1:50780+1:50 to 2:00
AB1IVT yield ranges (ng/uL)Example fragmentation durations for intestinal epithelium cell libraries (m:ss)20-20:00 to 0:2032-100:20 to 1:00410-401:00 to 1:30540-601:30 to 1:45660-801:40 to 1:50780+1:50 to 2:00
?. [Fragmentation]
After fragmentation immediately place all samples on the chilled 96 well aluminum block.
?. [Fragmentation]
Add 26 uL of Fragmentation Stop mix to each sample and transfer that sample to its pre-chilled 1.5 mL DNA LoBind tube.
For minimal sample loss Use a P200 pipette to add 26 uL of Fragmentation Stop mix to a single sample. Using the same pipette tip immediately transfer that sample to the 1.5 mL tube. Mix by pipetting until no mixing lines are observed. Repeat this for all samples (use a new pipette tip for each sample).
?. [Fragmentation]
Incubate the samples on ice for . At this time place aliquots of the 1x PrimeScript RT buffer, the  TruDrop PE2-N6 primer, and dNTPs   to start thawing.
on ice
?. [2nd Reverse Transcription]
If it has not been done yet, start preparing the following RT mastermix in a DNA LoBind Tube (only add the H2O and RT buffer):  AB1ReagentVolume Per Reaction2Ultrapure DNase/RNase-free H2O2 μL35x PrimeScript RT buffer4 μL4RNase OUT (40 U/μL)2 μL5PrimeScript RT enzyme (200 U/μL)1 μL6total9 μL
AB1ReagentVolume Per Reaction2Ultrapure DNase/RNase-free H2O2 μL35x PrimeScript RT buffer4 μL4RNase OUT (40 U/μL)2 μL5PrimeScript RT enzyme (200 U/μL)1 μL6total9 μL
Always mix the RT buffer by pipetting before using it or aliquoting it out. This should be done until no mixing lines are observed.
?. [Fragmentation]
Place each sample tube on a magnetic tube rack for  at  or until the beads have fully separated from the supernatant.
0 Room temperature
?. [Fragmentation]
Use a P200 pipette to carefully aspirate the supernatant without removing/disturbing the bead pellet.
While doing this, slowly pipette from the side of each tube that is opposite the bead pellet. Do not touch the beads with the pipette tip.
?. [Fragmentation]
Wash the Bead pellet with  ethanol twice at .
0 Room temperature
?. [Fragmentation]
For a single sample: Keeping the tube on the magnet rack gently wash the beads with  of  ethanol. Rotate each tube 180°, allowing the beads to re-pellet on the side now closest to the magnet.
400 µl
Pipette the ethanol onto the opposite side of the tube from the beads so as to not greatly disturb the bead pellet. If some beads do not pass from one side of the tube to the other, this can sometimes be remedied by taping on the side of the tube where the beads are stuck. For this AMPurification in particular speed is generally prioritized over passing all beads across the tube so one should proceed to step 65.2. If one would still like to have stuck beads pass across the tube then follow the instructions below.If this does not work it is recommended to use the beads that do pass to the other side of the tube to loosen the beads that are stuck by slowly moving the tube around on the magnet such that the beads that were stuck are closest to the magnet. At this point, slightly moving the tube up and down with some rotation can then sometimes dislodge the beads that are stuck. The tube can then be rotated 180° again to pass all other beads across the tube. If some beads are still stuck after this it is generally recommended to proceed anyways. It is not essential for all beads to pass across the tube though it is recommended.
?. [Fragmentation]
Repeat Step 65.1 for each sample.
?. [Fragmentation]
Again rotate each tube 180°, allowing the beads to re-pellet on the side now closest to the magnet. All tubes should now be in their original orientation.
?. [Fragmentation]
Use a pipette to slowly aspirate and discard the Ethanol for each sample. Draw the ethanol up from the opposite side of the bead pellet. Do not disturb the bead pellet with the pipette tip.
?. [Fragmentation]
Repeat steps 65.1 - 65.4 to for the second  ethanol wash.
?. [Fragmentation]
After the second ethanol wash, quick-spin the sample tubes in a mini-centrifuge for   at  to collect any residual ethanol at the bottom of the tube.
0 Room temperature
?. [Fragmentation]
Place the tubes back on the magnet rack and then use a P200 or smaller pipette to remove any residual ethanol from the bottom of the tube. Do not touch the bead pellet!
?. [Fragmentation]
Leaving the tubes open on the magnetic tube rack allow each bead pellet to air dry for  at . While samples are drying, do the following:Set up and start a thermal cycler with the following protocol:                             hold            Lid:   Pre-chill 1 PCR tube for each sample  . Use the 96 well aluminum block for the PCR tubes. If possible, add 1 uL of dNTP's with 2 uL of TruDrop PE2-N6 primer in the PCR tube (otherwise this can be done later).                hold            Lid: Pre-chill 1 PCR tube for each sample . Use the 96 well aluminum block for the PCR tubes.
0 Room temperature
65 °C
105 °C
on ice
Place a dust-free cover over the tubes to prevent dust from falling into them. All samples should be dry after 5 minutes. On rare occasions a sample may take 6 or 7 minutes to dry.Always mix the PE2-N6 primer and dNTP's by pipetting before using it or aliquoting it out. This should be done until no mixing lines are observed.
?. [Fragmentation]
Once a sample is dry add  RNA Elution buffer to just that dry sample and place on a normal tube rack at .
8 µl
0 Room temperature
?. [Fragmentation]
Gently flick the bottom of the tube to re-suspend the bead pellet in the RNA Elution buffer.
Avoid flinging the solution onto the top half of the tube. Avoid flinging the solution onto the inside of the lid. DO NOT VORTEX!
?. [Fragmentation]
Incubate the sample at  for  on a normal tube rack. During this time perform the following: finish adding  of dNTPs and  of TruDrop PE2-N6 primer to the pre-staged PCR tubes.  AB1ReagentVolume Per Reaction2Ultrapure DNase/RNase-free H2O2 μL35x PrimeScript RT buffer4 μL4RNase OUT (40 U/μL)2 μL5PrimeScript RT enzyme (200 U/μL)1 μL6total9 μL
0 Room temperature
1 µl
2 µl
AB1ReagentVolume Per Reaction2Ultrapure DNase/RNase-free H2O2 μL35x PrimeScript RT buffer4 μL4RNase OUT (40 U/μL)2 μL5PrimeScript RT enzyme (200 U/μL)1 μL6total9 μL
RNA/DNA will not elute if samples are kept cold during this step.Always mix the PE2-N6 primer, dNTP's, and RT buffer by pipetting before using it or aliquoting it out. This should be done until no mixing lines are observed.
?. [Fragmentation]
Spin the sample tubes in a  mini-centrifuge for   at  to collect everything at the bottom of the tube.
0 Room temperature
Spinning for longer than 2 seconds here can result in AMPure beads getting stuck at the bottom of the tube at step 51.
?. [Fragmentation]
Place the tube on the magnet rack for about   to pellet the beads on the side of the tube.
If not all of the beads pellet against the magnet that is okay. They can be removed in the following step.
?. [Fragmentation]
Use a P20 pipette to transfer the supernatant () of each sample to its respective chilled PCR tube. Pipette up and down (5x) to mix the sample with the primer and dNTPs.
8 µl
Carrying the magnetic AMPure beads forward should be avoided. Residual AMPure beads can be removed by pipetting the sample out onto the side of the 1.5mL DNA LoBind tube directly above the magnet. Slowly pipette up and down 2-3 times, and then allow the beads to cluster on the magnet for 2-3 seconds. Then slowly draw the supernatant back into that same pipette tip while being careful to avoid drawing up any beads that may be present.
?. [2nd Reverse Transcription]
Incubate all samples at  for  on the thermal cycler set up in step 68. During this time finish making the RT master mix (step 75, add the RNase OUT and PrimScript RT enzyme). Mix by pipetting.
65 °C
?. [2nd Reverse Transcription]
Remove all samples form the thermal cycler and cool on ice (use the chilled 96-well block for about 5-10 seconds) before adding 9 uL of RT master mix to each sample. Using the same pipette tip that was used to add the RT mix, mix the sample by pipetting.
?. [2nd Reverse Transcription]
Start a thermal cycler following the below protocol:         for          for          for             holdlid: Place the samples on the thermal cycler heat-block when it reaches . During this time prepare two 1.5 mL DNA LoBind tubes for each sample. Pre-chill one of the two . Keep the other one at room temperature. If more  ethanol will be needed now is a great time to prepare it.
30 °C
42 °C
70 °C
4 °C
105 °C
30 °C
on ice
During this time if the sample indices have not already been chosen for each sample that should be done here. The PCR reactions after the RT cannot be completed without knowing which samples will have which index pairs. For assistance in picking which indices can be used together it is recommended to follow the indexing guidelines in the Illumina document "index-adapters-pooling-guide-1000000041074-08" (specifically the section: "IDT for Illumina-TruSeq UD Indexes").Further indexing guidelines can be found in the Sequencing section (step 130) of this protocol.
?. [2nd Reverse Transcription]
Place all samples on the chilled 96 well block . Add  of Ultrapure DNase/RNase-free H2O to each sample.
on ice
30 µl
?. [2nd Reverse Transcription]
Use a P200 pipette to add  of Ampure XP beads to a single sample. Using the same pipette tip, transfer the sample to a room temperature 1.5 mL DNA LoBind Tube and pipette up and down until no mixing lines are observed. DO NOT VORTEX.
35 µl
?. [2nd Reverse Transcription]
Repeat step 93 for each sample.
?. [In Vitro Transcription (IVT)]
Leaving the tubes open on the magnetic tube rack allow each bead pellet to air dry for  at . While samples are drying get out 1 PCR tube and one 0.5 mL DNA LoBind tube for each sample (do not use strip tubes or plates) and pre-chill . Use the 96 well aluminum block for the PCR tubes.
0 Room temperature
on ice
Place a dust-free cover over the drying tubes to prevent dust from falling into them. All samples should be dry after 5 minutes. On rare occasions a sample may take 6 or 7 minutes to dry.
?. [In Vitro Transcription (IVT)]
Once a sample is dry, add  RNA Elution buffer to just that dry sample and place on a normal tube rack at .
20 µl
0 Room temperature
?. [In Vitro Transcription (IVT)]
Gently flick the bottom of the tube to re-suspend the bead pellet in the H2O.
Avoid flinging the solution onto the top half of the tube. Avoid flinging the solution onto the inside of the lid. DO NOT VORTEX!
?. [In Vitro Transcription (IVT)]
Incubate the sample at  for  on a normal tube rack. During this time prepare 1 Qubit tube for each sample, 2 Qubit tubes for standards, and one 2 mL tube for preparing the Qubit reagents.
0 Room temperature
RNA/DNA will not elute if samples are kept cold during this step.
?. [In Vitro Transcription (IVT)]
Spin the sample tubes in a  mini-centrifuge for   at  to collect everything at the bottom of the tube.
0 Room temperature
Spinning for longer than 2 seconds here can result in AMPure beads getting stuck at the bottom of the tube at step 51.
?. [In Vitro Transcription (IVT)]
Place the tube on the magnet rack for about   to pellet the beads on the side of the tube.
If not all of the beads pellet against the magnet that is okay. They can be removed in the following step.
?. [In Vitro Transcription (IVT)]
Use a P20 pipette to transfer the supernatant () of each sample to its respective chilled 0.5 mL DNA LoBind tube. Using the same pipette tip transfer  of the sample to it's respective chilled PCR tube. Repeat this process for the remaining samples. The PCR tube is now the sample tube. The 0.5 mL DNA LoBind tube is now the backup.
20 µl
9 µl
Carrying the magnetic AMPure beads forward should be avoided. Residual AMPure beads can be removed by pipetting the sample out onto the side of the 1.5mL DNA LoBind tube directly above the magnet. Slowly pipette up and down 2-3 times, and then allow the beads to cluster on the magnet for 2-3 seconds. Then slowly draw the supernatant back into that same pipette tip while being careful to avoid drawing up any beads that may be present.
?. [2nd Reverse Transcription]
Incubate each sample/bead mixture at  for .During this time do the following:If needed, prepare more  ethanol. Otherwise: start thawing the TruDrop PE1/PE2 indexed  primer mixes that will be used for each sample.
0 Room temperature
on ice
?. [2nd Reverse Transcription]
Place each sample tube on a magnetic tube rack for  at  or until the beads have fully separated from the supernatant. During this time do the following: start thawing the TruDrop PE1/PE2 indexed  primer mixes that will be used for each sample.
0 Room temperature
on ice
At the end of the 2 minutes the pellet can still look slightly diffuse. This is normal. Proceed to the next step anyways. The post-2nd-RT AMPurification can be very finicky. The diffuse nature of the beads is due to either the high glycerol concentration from the RT, or the large amount of protein from the RT, or both.
?. [2nd Reverse Transcription]
Carefully aspirate the supernatant without removing/disturbing the bead pellet.
While doing this, use a P200 pipette to slowly pipette from the side of each tube that is opposite the bead pellet. Do not touch the beads with the pipette tip. Do not aspirate any beads! If beads are removed by accident they can be recovered as follows:Hold the magnet rack such that the side of the tube at the top of the magnet is horizontal.Slowly pipette the entire solution out onto the top of the magnet making sure the supernatant does not contact the bead pellet. Slowly draw the supernatant up into the pipette, leaving behind any small amount of beads pulled out of solution on the side of the tube. If a 1-2 uL drop of supernatant is left behind that will not be a major problem.
?. [2nd Reverse Transcription]
Wash the Bead pellet with  ethanol twice at .
0 Room temperature
?. [2nd Reverse Transcription]
For a single sample: Keeping the tube on the magnet rack gently wash the beads with  of  ethanol. Rotate each tube 180°, allowing the beads to re-pellet on the side now closest to the magnet.
400 µl
Pipette the ethanol onto the opposite side of the tube from the beads so as to not greatly disturb the bead pellet. Then, rotate the tube. If some beads do not passing from one side of the tube to the other this can sometimes be remedied by taping on the side of the tube where the beads are stuck. If this does not work it is recommended to use the beads that do pass to the other side of the tube to loosen the beads that are stuck by slowly moving the tube around on the magnet such that the beads that were stuck are closest to the magnet. At this point, slightly moving the tube up and down with some rotation can then sometimes dislodge the beads that are stuck. The tube can then be rotated 180° again to pass all other beads across the tube. If some beads are still stuck after this it is generally recommended to proceed anyways. It is not essential for all beads to pass across the tube though it is recommended.During the first ethanol wash of the AMPurification after 2nd RT it is very normal for all of the beads to not pass across the tube when rotated on the magnet rack. The best way to avoid this is to rotate the tube ASAP after adding the first ethanol wash. It may take more tapping/moving/rotating the tube than normal to pass all beads across.
?. [2nd Reverse Transcription]
Repeat Step 97.1 for each sample.
?. [2nd Reverse Transcription]
Again rotate each tube 180°, allowing the beads to re-pellet on the side now closest to the magnet. All tubes should now be in their original orientation.
?. [2nd Reverse Transcription]
Use a pipette to slowly aspirate and discard the Ethanol. Draw the ethanol up from the opposite side of the bead pellet. Do not disturb the bead pellet with the pipette tip.
?. [2nd Reverse Transcription]
Repeat steps 97.1 - 97.4 to for the second  ethanol wash.
?. [2nd Reverse Transcription]
After the second ethanol wash, quick-spin the sample tubes in a  mini-centrifuge for   at  to collect any residual ethanol at the bottom of the tube.
0 Room temperature
?. [2nd Reverse Transcription]
Place the tubes back on the magnet rack and then use a P200 or smaller pipette to remove any residual ethanol from the bottom of the tube. Do not touch the bead pellet!
?. [2nd Reverse Transcription]
Leaving the tubes open on the magnetic tube rack, allow each bead pellet to air dry for   at . If a single 1.5 mL DNA LoBind tube has not yet been pre-chilled for each sample, prepare those now.
0 Room temperature
Place a dust-free cover over the drying tubes to prevent dust from falling into them. All samples should be dry after 2—2:30 minutes. At this step, it is generally recommended to just elute all samples here after 2 minutes. Allowing samples to over-dry here will result in the pellet becoming cracked very quickly, and them not being able to be fully re-suspended during elution.
?. [2nd Reverse Transcription]
Once a sample is dry, add  DNA Elution buffer to just that dry sample and place on a normal tube rack at .
40 µl
0 Room temperature
?. [2nd Reverse Transcription]
Incubate each sample/bead mixture at  for . While samples are eluting:Get out 1 PCR tube for each sample (do not use strip tubes or plates) and pre-chill . Use the 96 well aluminum block for the PCR tubes. Start thawing a 300 uL aliquot of 2x KAPA HiFi HotStart .
0 Room temperature
on ice
on ice
?. [2nd Reverse Transcription]
Place each sample tube on a magnetic tube rack for  at , or until the beads have fully separated from the supernatant.
0 Room temperature
For the RT Ampurification it is normal to have a thin film of beads that remain and continue to coat the bottom of the tube when placed against the magnet. As long as a distinct small pellet of beads is observed close to the magnet it is okay to proceed to step 106.
?. [2nd Reverse Transcription]
Gently flick the bottom of the tube to re-suspend the bead pellet in the DNA Elution buffer.
Avoid flinging the solution onto the top half of the tube. Avoid flinging the solution onto the inside of the lid. DO NOT VORTEX!
?. [2nd Reverse Transcription]
Spin the sample tubes in a  mini-centrifuge for   at  to collect everything at the bottom of the tube.
0 Room temperature
Spinning for longer than 2 seconds here can result in AMPure beads getting stuck at the bottom of the tube at step 51.
?. [2nd Reverse Transcription]
Use a P20 pipette to transfer the supernatant () of each sample to its respective chilled 1.5 mL DNA LoBind tube. Using the same pipette tip transfer  of the sample to it's respective chilled PCR tube. Repeat this process for the remaining samples. The PCR tube is now the sample tube. The 1.5 mL DNA LoBind tube is now the pre-PCR backup.
40 µl
20 µl
Carrying the magnetic AMPure beads forward should be avoided. Residual AMPure beads can be removed by pipetting the sample out onto the side of the 1.5mL DNA LoBind tube directly above the magnet. Slowly pipette up and down 2-3 times, and then allow the beads to cluster on the magnet for 2-3 seconds. Then slowly draw the supernatant back into that same pipette tip while being careful to avoid drawing up any beads that may be present.
This can be a pause point. It is highly recommended that all samples proceed with library preparation to finish preparing the final library. However, if necessary the tubes at this stage can be frozen at   until the library preparation can continue.
?. [Diagnostic qPCR]
Prepare the following qPCR mixture on ice:   AB1ReagentVolume per reaction2Ultrapure DNase/RNase-free H2O13 μL32x KAPA HiFi HotStart PCR Mix20 μL420x Eva Green Dye2 μL5Total35 μL
AB1ReagentVolume per reaction2Ultrapure DNase/RNase-free H2O13 μL32x KAPA HiFi HotStart PCR Mix20 μL420x Eva Green Dye2 μL5Total35 μL
?. [Diagnostic qPCR]
Decide and record which sample will be PCR'ed in which sample well.  It is recommended to space your samples so that none are located on the border of the plate. Example: We always use column 4 starting at row B and ending at row G. Add 35 uL of qPCR mixture to each well that will contain a sample.
?. [Diagnostic qPCR]
Mix the thawed 2x KAPA and all of the TruDrop primer mixes to be used by pipetting until no mixing lines are observed or at least by pipetting up and down 5x. Next, place a 96 well PCR Plate into a pre-frozen () PCR-Cooler rack.
-20 °C
?. [Diagnostic qPCR]
Add 4 uL of the TruDrop PE1/PE2 primer mixture that corresponds to each sample to the well of the PCR plate where that sample will be located.
?. [Diagnostic qPCR]
Add 1 uL of a single sample to it's corresponding well in the PCR plate. Make sure to pipette out into the liquid in each well and not onto the side of the plate.
?. [Diagnostic qPCR]
Repeat step 111 for each sample
?. [Diagnostic qPCR]
Seal the plate with a MicroSeal (BioRad MSB-100). Use the smooth side of the paper protector to press down around the edges of the wells containing the samples and around the edges of the plate.
?. [Diagnostic qPCR]
Spin the qPCr Plate at  to collect everything at the bottom of the well(s) and remove any bubbles.
Centrifuge: 600 33, 1 min, 4 10
?. [Diagnostic qPCR]
Set up a qPCR machine with the following protocol, load the plate in the machine and start the PCR machine:1)         for 2)         for 3)         for 4)         for 5) Go to 2) once (a total of two cycles)6)         for  7)         for 8)         for  with fluorescent read9) Go to 6) 23 times (a total of 24 cycles)Lid: Volume:
98 °C
98 °C
55 °C
72 °C
98 °C
65 °C
72 °C
105 °C
40 µl
This PCR normally takes about 1 hour 25 minutes. While the reaction is going the remaining sample (19.5 uL) PCR tubes, primer tubes, and KAPA should be placed on the chilled 96 well aluminum block and then stored in a clean 4oC fridge during the course of the reaction. This  is also a good point to replenish the ice in the ice bucket.
?. [Diagnostic qPCR]
Set the qPCR threshold within the exponential phase of amplification, but closer to where the signal starts to emerge from the noise to avoid the possibility of over amplification. We normally wil adjust to about 5000 RFU.
?. [Diagnostic qPCR]
Correct the resulting Ct value to account for differences in the amount of input material between the qPCR and library PCR. The library input is 19 times higher, and assuming a 100% PCR efficiency, the difference corresponds to log2 19 = 4.25 cycles. For example if the number of cycles determined by qPCR is 2 + 10.27, the required number of PCR cycles for library is 2 + (10.27 — 4.25) = 2 + 6.02 . This would then be rounded to 2 + 6 cycles. The rules we use for rounding here is that any number with a decimal value of 0.4 or more is rounded up to the nearest integer, while any number with a decimal value less that 0.4 is rounded down to the nearest integer. Example: 6.38 would be rounded down to 6 cycles, but 6.42 is rounded up to 7 cycles. Record this rounded value as the 2 + N, where N is the rounded adjusted number of cycles.
Expected result is normally 4-7 cycles depending on the sample.
?. [PCR Amplification]
Move the 96 well aluminum block with the samples, primer mixes, and KAPA back to the ice bucket.
?. [PCR Amplification]
Prepare the following PCR mixture :  AB1ReagentVolume per reaction2Ultrapure DNase/RNase-free H2O1 uL32x Kapa HiFi HotStart PCR mix25 uL4Total26 uL
on ice
AB1ReagentVolume per reaction2Ultrapure DNase/RNase-free H2O1 uL32x Kapa HiFi HotStart PCR mix25 uL4Total26 uL
?. [PCR Amplification]
To each sample add 5 uL of TruDrop PE1/PE2 primer mix to the corresponding sample tube.
?. [PCR Amplification]
To each sample add 26 uL of PCR mix to the corresponding sample tube. Mix the sample by pipetting until no mixing lines are observed.
?. [PCR Amplification]
Set up and start a PCR machine to follow the below protocol (wait to place the samples on the machine until the heat block is  ):1)         for 2)         for 3)         for 4)         for 5) Go to 2) once (a total of two cycles)6)         for  7)         for 8)         for  with fluorescent read9) Go to 6) N-1 times (a total of N adjusted cycles).10)          for 11) hold Lid: Volume: To the sample The value for N was determined as a part of step 116. If working with multiple libraries that require different numbers of cycles, set the thermal cycler to perform the highest number of cycles required, and manually remove individual libraries from the thermal cycler, when they reach the desired cycle number. For example, if library X requires 8 cycles, at the end of the 72°C step of cycle 8 pause the cycler and transfer library X to an ice bucket. Then, resume the PCR program. Once all the samples have undergone the required number of PCR cycles, transfer all tubes back to the thermal cycler for the final step, a 5 min incubation at 72°C. During the final 5 min extension at 72oC prepare 3 DNA LoBind tubes for each sample. 2 tubes will be kept at . The final tube should be chilled . To one of each sample's room temperature LoBind tubes add 20 uL of AMPure beads (part of this can be done later).
95 °C
98 °C
98 °C
55 °C
72 °C
98 °C
65 °C
72 °C
72 °C
4 °C
105 °C
50 µl
0 Room temperature
on ice
?. [PCR Amplification]
When the PCR is finished transfer all samples to the chilled 96-well aluminum block . Add  of DNA Elution buffer to the PCR product.
on ice
50 µl
?. [PCR Amplification]
To a single sample add 60 uL of AMPurebeads. Using the same pipette tip transfer the sample + bead mixture to its empty DNA LoBind tube. Mix by pipetting until no mixing lines are observed. DO NOT VORTEX.
This is the start of a double sided AMPurification. The lower bound is 0.6x (step 110). The upper bound is 0.8x (step 113).
?. [PCR Amplification]
Do step 123 for all other samples.
?. [PCR Amplification]
Incubate each sample/bead mixture at  for .During this time do the following:If needed, prepare more  ethanol.
0 Room temperature
?. [PCR Amplification]
Place each sample tube on a magnetic tube rack for  at  . It is important that samples are on the magnet rack for the full 2 minutes.
0 Room temperature
?. [PCR Amplification]
The finished library is what is in the supernatant. For a single sample, carefully transfer the supernatant to the second room temperature 1.5 mL DNA LoBind tube containing 20 uL of AMPure beads. Do not carry forward any beads. Using the same pipette tip, mix the sample by pipetting until no mixing lines are observed. The second tube is now the sample tube.
While doing this, use a P200 pipette to slowly pipette from the side of each tube that is opposite the bead pellet. Do not touch the beads with the pipette tip. Do not aspirate any beads! If beads are removed by accident they can be removed as follows:Close alltubes and hold the magnet rack such that the side of the tube at the top of the magnet is horizontal.Slowly pipette the entire solution out onto the top of the magnet making sure the supernatant does not contact the bead pellet. Slowly draw the supernatant up into the pipette, leaving behind any small amount of beads pulled out of solution on the side of the tube. If a 1-2 uL drop of supernatant is left behind that will not be a major problem.
?. [PCR Amplification]
Wash the Bead pellet with  ethanol twice at .
0 Room temperature
?. [PCR Amplification]
For a single sample: Keeping the tube on the magnet rack gently wash the beads with  of  ethanol. Rotate each tube 180°, allowing the beads to re-pellet on the side now closest to the magnet.
400 µl
Pipette the ethanol onto the opposite side of the tube from the beads so as to not greatly disturb the bead pellet. Then, rotate the tube. If some beads do not passing from one side of the tube to the other this can sometimes be remedied by taping on the side of the tube where the beads are stuck. If this does not work it is recommended to use the beads that do pass to the other side of the tube to loosen the beads that are stuck by slowly moving the tube around on the magnet such that the beads that were stuck are closest to the magnet. At this point, slightly moving the tube up and down with some rotation can then sometimes dislodge the beads that are stuck. The tube can then be rotated 180° again to pass all other beads across the tube. If some beads are still stuck after this it is generally recommended to proceed anyways. It is not essential for all beads to pass across the tube though it is recommended.
?. [PCR Amplification]
Repeat Step 117.1 for each sample.
?. [PCR Amplification]
Again rotate each tube 180°, allowing the beads to re-pellet on the side now closest to the magnet. All tubes should now be in their original orientation.
?. [PCR Amplification]
Use a pipette to slowly aspirate and discard the Ethanol. Draw the ethanol up from the opposite side of the bead pellet. Do not disturb the bead pellet with the pipette tip.
?. [PCR Amplification]
Repeat steps 117.1 - 117.4 to for the second  ethanol wash.
?. [PCR Amplification]
After the second ethanol wash, quick-spin the sample tubes in a  mini-centrifuge for   at  to collect any residual ethanol at the bottom of the tube.
0 Room temperature
?. [PCR Amplification]
Place the tubes back on the magnet rack and then use a P200 or smaller pipette to remove any residual ethanol from the bottom of the tube. Do not touch the bead pellet!
?. [PCR Amplification]
Leaving the tubes open on the magnetic tube rack allow each bead pellet to air dry for   at . If a single 1.5 mL DNA LoBind tube has not yet been pre-chilled for each sample, prepare those now.
0 Room temperature
Place a dust-free cover over the drying tubes to prevent dust from falling into them. All samples should be dry after 2—2:30 minutes.
?. [PCR Amplification]
Once a sample is dry, add  DNA Elution buffer to just that dry sample and place on a normal tube rack at .
20 µl
0 Room temperature
?. [PCR Amplification]
Gently flick the bottom of the tube to re-suspend the bead pellet in the DNA Elution Buffer.
Avoid flinging the solution onto the top half of the tube. Avoid flinging the solution onto the inside of the lid. DO NOT VORTEX!
?. [PCR Amplification]
Incubate each sample/bead mixture at  for .
0 Room temperature
?. [PCR Amplification]
Spin the sample tubes in a  mini-centrifuge for   at  to collect everything at the bottom of the tube.
0 Room temperature
Spinning for longer than 2 seconds here can result in AMPure beads getting stuck at the bottom of the tube at step 51.
?. [PCR Amplification]
Place each sample tube on a magnetic tube rack for  at , or until the beads have fully separated from the supernatant.
0 Room temperature
For the RT Ampurification it is normal to have a thin film of beads that remain and continue to coat the bottom of the tube when placed against the magnet. As long as a distinct small pellet of beads is observed close to the magnet it is okay to proceed to step 106.
?. [PCR Amplification]
Use a P20 pipette to transfer the supernatant () of each sample to its respective chilled 1.5 mL DNA LoBind tube. This is the final library.
20 µl
Carrying the magnetic AMPure beads forward should be avoided. Residual AMPure beads can be removed by pipetting the sample out onto the side of the 1.5mL DNA LoBind tube directly above the magnet. Slowly pipette up and down 2-3 times, and then allow the beads to cluster on the magnet for 2-3 seconds. Then slowly draw the supernatant back into that same pipette tip while being careful to avoid drawing up any beads that may be present.
?. [PCR Amplification]
Use 2 uL from the sample to measure the DNA concentration of the sample via Qubit using the Qubit DNA HS assay kit.
Sample Concentration should be between 1-3 ng/uL.
?. [PCR Amplification]
Repeat step 126 for all remaining samples.
?. [PCR Amplification]
Incubate each sample/bead mixture at  for  on a normal tube rack.
0 Room temperature
?. [PCR Amplification]
Place each sample tube on a magnetic tube rack for  at  . It is important that samples are on the magnet rack for the full 2 minutes.
0 Room temperature
?. [PCR Amplification]
The sample is now what is on the beads. Carefully aspirate the supernatant without removing/disturbing the bead pellet.
While doing this, use a P200 pipette to slowly pipette from the side of each tube that is opposite the bead pellet. Do not touch the beads with the pipette tip. Do not aspirate any beads! If beads are removed by accident they can be recovered as follows:Hold the magnet rack such that the side of the tube at the top of the magnet is horizontal.Slowly pipette the entire solution out onto the top of the magnet making sure the supernatant does not contact the bead pellet. Slowly draw the supernatant up into the pipette, leaving behind any small amount of beads pulled out of solution on the side of the tube. If a 1-2 uL drop of supernatant is left behind that will not be a major problem.
?. [PCR Amplification]
Samples should be stored at  until they can be sequenced.
-80 °C
?. [Sequencing]
Quantify samples via BioAnalyzer and qPCR prior to sequencing.
?. [Sequencing]
At this point the libraries should be treated like normal TruSeq libraries. Use normal TruSeq sequencing primers.Libraries have been succesfully sequenced on NovaSeq with the following settings:Read type: Paired- EndRead 1: 50 cycles or more (Cel Barcodes + UMI)Read 2: 50 cycles or more (we normally use at least 100 cycles for transcript)i7 index read: 8 cyclesi5 index read: 8 cycles
?. [Sequencing]
If sequencing TruDrop libraries in pools alongside higher percentages of other Illumina compatible libraries (70% of the total pool or more is from other Illumina compatible libraries), then one can use each pair of indexes (one i7, and one i5) once per sequencing lane per sequencing run. There are no other limitations on sequencing index usage, provided that a library can be made from the index pair (do not use pair UDI0009). Example: UDI0025 could be used with UDI0001. So long as the indices aren’t repeated across multiple libraries in a single sequencing pool. This option is recommended so as to not run out of certain primer pair before others.Disclaimer: Our operating range is 70% at the lowest for the above cases, but I suspect that 50% may also work if necessary. If you are unsure, it is recommended that you consult with your sequencing core/provider on which index pairs to use. If consultation is not possible, you can always follow the criteria for library pooling with 17-24 TruDrop libraries, in that order, and it should work every time. The downside to this option is that certain index primers will be expended at a significantly higher rate resulting in varying turnover rates for index pairs.If Sequencing 1-8 TruDrop libraries alongside lower percentages of other illumina compatible libraries (70% of the total pool or less is from other illumina compatible libraries) then follow the bellow pooling guidelines:Use UDI0001, then UDI0002, then UDI0003, then UDI0004, then UDI0005, then UDI0006, then UDI0007, then UDI0008,Or Use UDI0017, then UDI0018, then UDI0019, then UDI0020, then UDI0021, then UDI0022, then UDI0023, then UDI0024,If Sequencing 9-16 TruDrop libraries alongside lower percentages of other illumina compatible libraries (70% of the total pool or less is from other illumina compatible libraries) then follow the bellow pooling guidelines:Use UDI0001, then UDI0002, then UDI0003, then UDI0004, then UDI0005, then UDI0006, then UDI0007, then UDI0008, then UDI0017, then UDI0018, then UDI0019, then UDI0020, then UDI0021, then UDI0022, then UDI0023, then UDI0024,Or Use UDI0017, then UDI0018, then UDI0019, then UDI0020, then UDI0021, then UDI0022, then UDI0023, then UDI0024, then UDI0001, then UDI0002, then UDI0003, then UDI0004, then UDI0005, then UDI0006, then UDI0007, then UDI0008,If Sequencing 17-24 TruDrop libraries alongside lower percentages of other illumina compatible libraries (70% of the total pool or less is from other illumina compatible libraries) then follow the bellow pooling guidelines:Use UDI0001, then UDI0002, then UDI0003, then UDI0004, then UDI0005, then UDI0006, then UDI0007, then UDI0008, then UDI0017, then UDI0018, then UDI0019, then UDI0020, then UDI0021, then UDI0022, then UDI0023, then UDI0024, then UDI0010, then UDI0011, then UDI0012, then UDI0013, then UDI0014, then UDI0015, then UDI0016, then UDI0025Or Use UDI0017, then UDI0018, then UDI0019, then UDI0020, then UDI0021, then UDI0022, then UDI0023, then UDI0024, then UDI0001, then UDI0002, then UDI0003, then UDI0004, then UDI0005, then UDI0006, then UDI0007, then UDI0008, then UDI0010, then UDI0011, then UDI0012, then UDI0013, then UDI0014, then UDI0015, then UDI0016, then UDI0025