May 20, 2025

Public workspaceTIRTL-seq 384-well

DOI
dx.doi.org/10.17504/protocols.io.eq2lyx37wgx9/v1
  • 1St. Jude Children's Research Hospital
Mikhail Pogorelyy
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DOI: dx.doi.org/10.17504/protocols.io.eq2lyx37wgx9/v1
External link: https://www.biorxiv.org/content/10.1101/2024.09.16.613345v2
Protocol Citation: Mikhail Pogorelyy, Allison Kirk, Samir Adhikari, Balaji Sundararaman, Paul Thomas 2025. TIRTL-seq 384-well. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyx37wgx9/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 28, 2025
Last Modified: May 20, 2025
Protocol Integer ID: 125695
Keywords: immune repertoire sequencing, TCR, T cell receptor
Funders Acknowledgements:
NIH
Grant ID: R01 AI136514
NIH
Grant ID: U01 AI144616
NIH
Grant ID: P01 AI165077
NIH
Grant ID: 75N93019C00052
NIH
Grant ID: 75N93021C00016
ALSAC at St. Jude
Grant ID: TIRTL Blue-sky
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Abstract
TIRTL-seq is a method to obtain paired TCR-sequencing information from millions of human T cells. It is based on splitting cells into 96-384 wells, preparing and sequencing TCRalpha and TCRbeta libraries from each well in miniaturized reactions, and then pairing alpha and beta chains based on co-occurrence patterns or relative frequency variations between wells. For more details about the method, please see our preprint (https://pubmed.ncbi.nlm.nih.gov/39345544/ ). The 384-well protocol requires some automation: a non-contact liquid dispenser (we have tested Dispendix I.Dot mini/S/L and Formulatrix Mantis) and a device to transfer 1 uL between 384-well plates (we use Integra Viaflo, but many other solutions should work equally well). We have also designed a 96-well protocol with increased volumes, which does not require any automation but has lower resolution.



Schematic of TIRTL-seq protocol. Briefly, a cell suspension is distributed into 384-well plates containing RT/lysis mastermix under a hydrophobic overlay using non-contact liquid dispensers. After the RT reaction, PCR I mastermix with V-segment and C-segment primers is dispensed into the same plate. The PCR I product is then diluted and transferred to the PCR II plate for indexing PCR with well-specific unique dual indices. The PCR II products are pooled by centrifugation, purified, size-selected using magnetic beads, and sequenced on an Illumina platform. Total library preparation cost is listed for one 384-well plate.
Guidelines
Input material:
  1. We found the TIRTL-seq protocol to be robust and perform well on both fresh and cryopreserved PBMCs, as well as on T cells enriched by magnetic beads or sorted using a cell sorter. Sample viability should be >50%. If dealing with low viability (<50%) or low T cell content tissue, we recommend enriching for T cells (i.e. CD4/CD8 positive isolation with Dynabeads with Detach or sorting) prior to TIRTL-seq. Note that this version of the protocol works only for human T cells.
  2. Increasing the number of T cells per well increases the yield of paired TCRs without affecting the cost, with best results achieved at ~10 million PBMCs/purified T cells per 384-well plate (or 25K cells/well). If you have even more T cells available, it is recommended to do multiple plates per sample instead of scaling up volumes or loading more cells per well. Loading more cells per well (e.g. 50K cells/well or more) does not improve yield of unique clones.
  3. Note that TIRTL-seq is not able to pair clones found in <3 cells in your sample. If you have very few T cells, consider single cell/well sorting modification of the protocol (see Appendix I; almost all individual cells will be paired in this modification) or doing antigen-independent T cell expansion before running the TIRTL-seq to expand small clones to >3 cells/clone level.
Materials
Download TIRTL_seq_primers_v1.xlsTIRTL_seq_primers_v1.xls
Primers (see Excel file above for sequences)
  1. hTCRab_Vmix_P7 (Forward V multiplex with P7 adapters)
  2. hTCRab_Cmix_IDXX_P5 (Reverse C-segment primers with P5 adapters and plate barcodes)
  3. Well Index Primers (Nextera P5 and P7 with 10nt indices from cite SS3xpress protocol with minor modifications)

Reagents
Cell culture
ReagentRPMI-1640Thermo Fisher ScientificCatalog #22400089
ReagentFetal Bovine SerumThermo Fisher ScientificCatalog #16140071
ReagentL-Glutamine (200 mM)Thermo FisherCatalog #25030081
ReagentPenicillin-Streptomycin (10,000 U/mL)Thermo Fisher ScientificCatalog #15140122
ReagentGibco™ DPBS no calcium no magnesiumThermo Fisher ScientificCatalog #14190144

Molecular Biology
ReagentVapor-LockQiagenCatalog #981611
ReagentTriton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #X100-100ML
ReagentNuclease-Free Water (not DEPC-Treated)Thermo Fisher ScientificCatalog #AM9937
ReagentdNTP Mix (dATP, dCTP, dGTP, and dTTP, each at 10mM) Thermo Fisher ScientificCatalog #R0192
ReagentRandom HexamerThermo Fisher ScientificCatalog ##SO142
ReagentOligo(dT)18Integrated DNA Technologies, Inc. (IDT)Catalog #custom order
ReagentDithiothreitol (DTT)Thermo Fisher ScientificCatalog #707265ML
ReagentRecombinant RNase InhibitorTakara Bio Inc.Catalog #2313B
ReagentMaxima™ H Minus Reverse TranscriptaseThermo Fisher ScientificCatalog #EP0753
Reagent2X KAPA2G Fast Multiplex KitRoche DiagnosticsCatalog #7961430001
ReagentQ5 Hot Start High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0493L
Reagent Ampure XP beads Beckman CoulterCatalog #A63881
ReagentQubit® dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854
ReagentD1000 ScreenTapeAgilent TechnologiesCatalog #5067-5582
ReagentD1000 ReagentsAgilent TechnologiesCatalog #5067-5583
ReagentD1000 LadderAgilent TechnologiesCatalog #5067-5586

Plastics and Other Consumables
ReagentArmadillo PCR Plate, 384-well, clear, clear wellsThermo FisherCatalog #AB2384
ReagentMicroAmp™ Clear Adhesive FilmThermo Fisher ScientificCatalog #4306311 (For sealing plates for PCR and storage)
ReagentSealing films PolyesterVWR International (Avantor)Catalog #60941-062 (For sealing VaporLock plates and other steps where a loose seal is desired, e.g. transferring plates between pre- and post-PCR I zones)
Reagent12.5 μL GRIPTIPS (Sterile, Filter, XYZ Racks of 384 Tips)Integra BiosciencesCatalog #6455
ReagentV-bottom 200 mL reservoirClickBio, Inc.Catalog #CBVBLOK200-1


Recommended Equipment
384-well plate thermocyclers: We use Bio Rad C1000 Touch/S1000, but almost any other thermocycler should work.

Plate centrifuges with bucket rotors: We use a Sorvall ST8R/ST8 with an M10 rotor. One is in the pre-PCR zone, one is in the post-PCR zone (for pooling by centrifugation). NOTE: for pooling by centrifugation with ClickBio reservoir, make sure it fits into the bucket (for Thermo Fisher M10 rotor it fits into unsealed bucket, cat. 75005723).

Dispendix I.Dot Mini, I.Dot S, or I.Dot L: Used for dispensing cells and Lysis+RT master mix. Can also be used to dispense PCR I master mix, and PCR II master mix.

Formulatrix Mantis: Used for dispensing PCR I and PCR II master mix. Can also be used to dispense Lysis+RT master mix and cells (with appropriate decontamination).

Integra Viaflo (384-channel, 12.5 uL head) or Integra Mini 96 (12.5 uL): Used for dispensing VaporLock (pre-PCR zone) and Well Index primers (post-PCR I zone). Can also be used for diluting PCR I product (post-PCR I zone). Separate instruments will be needed for each zone.

Integra WellJet: Used for diluting PCR I product, can also be used to dispense PCR II master mix.

Thermo Fisher Qubit Fluorometer: Used to quantify the final library.

Agilent Tapestation: Used to determine library size and quality.
Safety warnings
To avoid contamination with PCR products it is crucial to separate pre-PCR and post-PCR zones. Reagents, equipment, and samples should never move from post-PCR zones to pre-PCR zones. We recommend doing all work in PCR workstations or biosafety cabinets and using UV to decontaminate the workspace after each procedure. We also recommend that you work in gloves, use filtered tips, and put away primers, beads, and other reagents before opening any PCR product. Steps performed in the pre-PCR zone:
PBMC/CD4/CD8 isolation, RT reaction setup, PCR I reaction setup, primer dilution, reagent/primer storage. PCR I/PCR II plates are never opened or stored in this zone. Steps performed in the post-PCR zone: PCR I reaction dilution, transfer of PCR I product to PCR II reaction. PCR II pooling, PCR II pool size-selection, and QC. In our lab, PCR II pooling, size-selection, and QC (the stage with highest PCR product concentration) is also done in a third separate room (post PCR II zone).
Ethics statement
Please note that this protocol is designed for human samples and prior approval from your Institutional Review Board (IRB) or equivalent ethics committee is required before commencing any work. Please make sure that processing of human tissues is performed in accordance with your local biosafety regulations, including appropriate containment and personal protective equipment.
Before start
Preparation of Vapor Lock Overlay Plates
Load 3 uL/well Qiagen VaporLock (or similar) to each well of a 384-well plate using an Integra Viaflo or Integra Mini. Loosely seal each plate. Plates loaded with VaporLock can be stored at room temperature indefinitely and therefore can be prepared in advance in large quantities.

Primer Preparations
PCR I - Forward primer mix (hTCRab_Vmix_P7 2.27uM each):
We use a subset of forward V-segment specific primers from Howie et al (Sci Trans Med, 2015: https://www.science.org/doi/full/10.1126/scitranslmed.aac5624) modified with P7 adapter sequences and excluding pseudogene-specific primers. 88 primers are ordered from IDT in a 96-well plate format at 400 uM each in IDTE buffer. See the attached excel file for list of primer sequences.
  1. Mix equal volume of each primer with multichannel pipette in a reservoir.
  2. Aliquot 400 uL of primer mix into 1.5 ml tubes with 400 uL of nuclease free water to get 2.27uM each working dilution.
  3. Store tubes at -20 °C.

PCR I - Reverse primer mixes with plate-specific barcodes (hTCRab_Cmix_IDXX_P5 10uM each): We included 7 or 10nt long custom inline index sequences for pooling plates together for sequencing; these indices are distinct from well-specific barcodes in PCR II primers (see below). We chose the index sequences to reduce amplification bias among the different C-segment genes. Reverse primers with plate indices were ordered from IDT in tube format as 100 uM LabReady solution in IDTE buffer. See the attached excel file for list of primer sequences.
  1. Mix 50 uL of TCRalpha + 50 uL of TCRbeta + 400 uL of nuclease free water to get 10 uM each working dilution. Note the plate index, as this will be crucial for the demultiplex pipeline. We advise to mix TCRalpha_ID01 with TCRbeta_ID01, TCRalpha_ID02 with TCRbeta_ID02 and so on (other combinations are likely to work, but this has not been tested).
  2. Store tubes with mixed plate index reverse primers at -20 °C.

PCR II (indexing with well-specific barcodes):
We use primers from Hagemann-Jensen et al (Smart-seq3xpress - Nature Biotech, 2022: https://www.nature.com/articles/s41587-022-01311-4) with small modifications to a few underperforming barcodes. The primers were ordered from IDT in 384-well plate format 200 uM concentration (one plate of 384 i5 forward primers and one plate of 384 i7 reverse primers). See the attached excel file for list of primer sequences.

To minimize potential contamination and freeze-thaw cycles we recommend making as many mixed primer plates as possible and using a serial dilution strategy to optimize storage.

To make 20X stock (20uM each):
  1. Dispense 24 uL of nuclease-free water into 384 well plates and briefly centrifuge the plates.
  2. Using Integra Viaflo with 384-channel head: Dispense 3 uL of source forward primers (IDT 384 well plate, 200 uM each) from i5 IDT plate to the 384 well plates containing 24 uL of nuclease-free water using repeat dispense program (if making multiple plates). Eject tips once done.
  3. Dispense 3 uL of source reverse primers (IDT 384 well plate, 200 uM each) from i7 IDT plate to the 384 well plates containing 27 uL of diluted i5 primer using repeat dispense program.
  4. Seal the plates, making sure all the corners are sealed. Label the plates. (20 uM each of forward and reverse primers in 30 uL).
  5. Store the plates in -20 °C.

To make 1X working dilution primer plate (1 uM each) from 20X stock:
  1. Dispense 23.8 uL of nuclease-free water into 384 well plates using Integra Welljet liquid dispenser and briefly centrifuge the plates.
  2. Briefly centrifuge the Stock (20X) PCR II Primer plate (thaw first and centrifuge if frozen), carefully remove the seal.
  3. Mix the contents using the mix program in the Integra Viaflo. Discard the tips if there are some extra droplets outside or some air bubbles trapped.
  4. Stamp 1.25 uL from Stock (20X) PCR II Primer plate to the 384 well plates containing 23.8 uL of nuclease-free water using repeat dispense program (if making multiple plates).
  5. Seal the plates, making sure all the corners are sealed. Label the plates. (1 uM each of forward and reverse primers in 25 uL)
  6. Store the plates in -20 °C.



1. Cell Preparation (~1 hour)
1. Cell Preparation (~1 hour)
NOTE: This section describes processing for cryopreserved PBMCs/T cells. If starting from fresh/sorted/magnetically enriched PBMCs/T cells, go to step 3.
Thaw cryopreserved cells in a 37 °C water bath until only a small ice crystal remains, then add suspension drop-wise to 10 mL complete RPMI (cRPMI) pre-warmed to 37 °C.
  • Complete RPMI = RPMI 1640, 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin/streptomycin
Centrifuge 500xg for 5 minutes.
Aspirate supernatant and resuspend cells in 5 mL DPBS.
Count cells. We recommend counting cells with live/dead dyes (AO/PI, trypan blue, etc.) to determine cell viability.
Centrifuge 500xg for 5 minutes.
Aspirate supernatant and resuspend cells in 500 uL DPBS. Transfer immediately to a 1.5 mL tube, being careful to collect the entire volume.
  • If cell numbers are low, cells can also be resuspended in 150 uL DPBS and transferred to a 0.2 mL strip tube so that the cell pellet will be easier to see.
Centrifuge 500xg for 5 minutes.
  • If using 0.2 mL strip tubes, place the strip tubes in a strip tube rack and centrifuge using a plate bucket.
Aspirate supernatant carefully and resuspend to a final volume of exactly 110 uL DPBS.

CRITICAL: Do not alter this cell resuspension volume; always use exactly 110 uL for a full 384-well plate. If using only part of a 384-well plate for one sample, scale down volume accordingly; e.g. if using 192 wells, use 55 uL for resuspension; if using 96 wells, use 28 uL for resuspension, etc.
Critical
2. Reverse Transcription (~30 minutes setup, ~1.5 hours RT)
2. Reverse Transcription (~30 minutes setup, ~1.5 hours RT)
Prepare Lysis+RT master mix as follows:
ABC
Component1X Volume (1 well)430X Volume (1X 384-well plate)
Nuclease-Free Water 0.15 uL64.50 uL
Triton X-100 (1% v/v solution)0.04 uL17.20 uL
dNTPs (10 mM each)0.025 uL10.75 uL
Random Hexamer (200 ng/uL)0.00625 uL2.69 uL
Oligo(dT) (50 uM)0.025 uL10.75 uL
5X MaximaH RT Buffer0.1 uL43.00 uL
DTT (100 mM)0.025 uL10.75 uL
RNase Inhibitor (40 U/uL)0.02 uL8.60 uL
Maxima H Enzyme (200 U/uL)0.01 uL4.30 uL
TOTAL0.4 uL172.00 uL

Load 0.4 uL/well Lysis+RT master mix to each well of a 384-well VaporLock plate using Dispendix I.Dot L.
CRITICAL: Pulse centrifuge unsealed plate 1000xg for 10 seconds. Confirm Lysis+RT mix volume is visible below the VaporLock layer for all wells. Sealing the plate before centrifugation may cause reagent loss (and ultimately sequencing failure) for some wells due to reagent droplets sticking to the seal.
Critical
Load 0.25 uL/well cells to the 384-well plate using Dispendix I.Dot L.

NOTE:
  • Each plate can be divided according to experimental needs, e.g. rows A-H for one sample, rows I-P for another.
  • Cells can also be sorted directly into 384-well plates—see Appendix I.
  • This protocol has been tested and works well for 125-500 nL of cell suspension/well. The cell suspension volume can be slightly adjusted depending on your non-contact liquid dispenser.
CRITICAL: Pulse centrifuge unsealed plate 1000xg for 10 seconds. Confirm RT+lysis mix and cell volume is visible below the VaporLock layer for all wells.
Critical
Seal plate and pulse centrifuge again 1000xg for 10 seconds. Proceed to RT reaction immediately.
Incubate in a thermocycler with the following protocol:
AB
TemperatureTime
42 °C5 minutes
25 °C10 minutes
50 °C60 minutes
94 °C5 minutes
4 °Chold
SAFE STOP POINT: The RT plate can safely be stored at -80 °C for several days before proceeding to PCR I.
3. PCR I (~30 minutes setup, ~1 hour PCR)
3. PCR I (~30 minutes setup, ~1 hour PCR)
In pre-PCR zone:
Prepare PCR I master mix as follows:
ABC
Component1X Volume (1 well)430X Volume (1X 384-well plate)
Nuclease-Free Water 0.425 uL182.8 uL
2X KAPA2G Fast Multiplex1.25 uL537.5 uL
hTCRab_Vmix (2.27 uM each)0.225 uL96.8 uL
hTCRab_Cmix_IDXX_P5 (10 uM each)0.1 uL43.0 uL
TOTAL2.0 uL860.0 uL
CRITICAL: If multiple plates will be pooled and sequenced together in the same run, each plate MUST use a unique Plate Index Reverse primer. Note the Plate Index primer used for each plate, and prepare the master mix for each plate separately.
Dispense 2 uL/well PCR I master mix to the RT 384-well plate using Dispendix I.Dot L or Formulatrix Mantis.
Pulse centrifuge unsealed plate 1000xg for 10 seconds.
Seal the plate and pulse the centrifuge again 1000xg for 10 seconds.
Incubate in a thermocycler with the following protocol:
ABC
TemperatureTimeCycles
95 °C3 minutes
95 °C15 seconds20 cycles
59 °C30 seconds
72 °C1 minute
72 °C5 minutes
4 °Chold
SAFE STOP POINT: The PCR I plate can safely be stored at -20 °C for days to a few weeks before proceeding to PCR II.
PCR
4. PCR II (~45 minutes setup, ~45 minutes PCR)
4. PCR II (~45 minutes setup, ~45 minutes PCR)
In pre-PCR zone:
Prepare Q5 PCR II master mix as follows:
ABC
Component1X Volume (1 well)430X Volume (1X 384-well plate)
Nuclease-Free Water 1.85 uL833.0 uL
5X Q5 Buffer1.00 uL450.0 uL
dNTPs (10 mM each)0.10 uL45.0 uL
Q5 Hot Start DNA Polymerase0.05 uL22.5 uL
TOTAL2.0 uL860.0 uL

Load 3 uL/well Q5 master mix in an empty 384-well VaporLock plate using Dispendix I.Dot L or Formulatrix Mantis.
Loosely seal plate and pulse centrifuge 1000xg for 10 seconds.
Transfer plate to post-PCR I zone.

CRITICAL: To prevent contamination between experiments, it is critical to not open the PCR I plate in the pre-PCR I zone; it should only be opened in the post-PCR I zone. Similarly, DO NOT return to the pre-PCR zone on the same day after proceeding past this step.
Critical
In post-PCR I zone:

CRITICAL: To prevent contamination of primer plates, open the primer plate in the PCR workspace first, and reseal and store the primer plate BEFORE opening any PCR I product plates. DO NOT simultaneously have the primer plate and diluted PCR I products opened in the PCR workspace.
Critical
Add 1 uL/well Well Indexing primers (1 uM each) from 1X primer plate to Q5 PCR II master mix plate using Integra Viaflo.
Reseal 1X primer plate and return to -20 °C storage.
Dilute PCR I product ~10X by adding 18 uL nuclease-free water to PCR I plate using Integra WellJet.
Add 1 uL diluted PCR I product to Q5+Primer plate using Integra Viaflo.

NOTE: Dilution of PCRI product can also be performed without the WellJet: dilute PCR I product by adding 12 uL of water with Integra and transfer 0.75 uL to PCR II plate.
Seal plate and pulse centrifuge 1000xg for 10 seconds.
Incubate in a thermocycler with the following protocol:

CRITICAL: Choose the number of PCR cycles based on the number of cells loaded in the experiment. For experiments with >2000 cells/well, 15 cycles is recommended. For experiments with 1-2000 cells/well, 16-18 cycles is recommended.
ABC
TemperatureTimeCycles
98 °C30 seconds
98 °C10 seconds15-18 cycles
58 °C10 seconds
72 °C50 seconds
72 °C2 minutes
4 °Chold
PCR
Proceed to pooling and cleanup.

CRITICAL: Proceed immediately to pooling and cleanup. PCR II products cannot be safely stored for more than a few hours without cleanup.
Critical
5. PCR II Pooling and Cleanup (~45 minutes)
5. PCR II Pooling and Cleanup (~45 minutes)
In post-PCR II zone:
Pool PCR II products by carefully inverting the PCR II plate into a ClickBio V-bottom 200 mL reservoir. Ensure the plate fits snugly in the reservoir.
Centrifuge the plate+reservoir assembly at 100xg for 30 seconds, such that centrifugation will empty all wells into the reservoir. After centrifugation, ensure all wells in the plate are empty.
Collect 100 uL pooled PCR II product and transfer to a clean 1.5 mL tube.

CRITICAL: Avoid collecting the VaporLock layer that floats on top of the PCR II product. Approximately 1.8 mL PCR II product will be present in the reservoir.
Critical
Purify the PCR II library using AMPure beads at ratio of beads to sample indicated below:

  • For most samples, we recommend using a 0.9:1 bead:sample ratio.

  • If using high numbers of cells (>10,000 cells/well or 4 million cells/plate), using a 0.8:1 bead:sample ratio may yield a cleaner Tapestation trace.

  • If performing TIRTL-seq on single cells directly sorted into plates, use a 0.7:1 bead:sample ratio and perform the full cleanup (using the same ratio of beads:sample) twice.
Mix beads and PCR II products well and incubate for 10 minutes at room temperature.
Place beads on a magnet and wait for the solution to clear (~3 minutes).
Carefully remove supernatant without disturbing beads.
Wash beads with 200 uL freshly prepared 80% ethanol. Wait for 30 seconds, then discard the ethanol.
Repeat the above wash step for 2 washes total.
Briefly spin the tube, remove residual ethanol and air dry beads for 1-3 minutes.
Elute final library by adding 45 uL Nuclease-free water to beads and mixing well. Incubate for 5 minutes at room temperature.
Place beads on a magnet and wait for the solution to clear (~3 minutes).
Carefully remove 45 uL purified library and proceed to final QC.
NOTE: Cleanup can also be automated using an Opentrons OT-2 robot or similar liquid handlers.
SAFE STOP POINT: Purified libraries can be stored at -20 °C for many days and weeks before sequencing.
Pause
6. Final Library QC (~15 minutes)
6. Final Library QC (~15 minutes)
In post-PCR II zone:
Quantify the final library using a Qubit fluorometer and the Qubit dsDNA HS assay according to manufacturer instructions. Expected concentration after purifying 100 uL of PCRII products eluted in 45 uL is >10 ng/uL, most likely ~30-40 ng/uL for samples with good viability and high number of cells/well.
Run the final library on an Agilent Tapestation using D1000 reagents (or similar) to inspect quality and confirm the library size. The final library size should be ~340nt, and no shorter (<250nt) products should be present.

A representative Tapestation D1000 trace is below.


7. Sequencing
7. Sequencing
Final purified libraries are ready to sequence on any Illumina sequencer (preferred read length 2x150nt).
The target read number is approximately 100 million reads per 10 million cells (or 100 million reads/per plate loaded with a maximum amount of cells).
If libraries are prepared from multiple plates that have been uniquely indexed in PCR I, and all libraries are of similar size and quality, they can be pooled (usually in equal proportion by mass) for sequencing in a single run in the same lane. Scale up the number of reads requested accordingly. Note that wells within plates are barcoded on both sides, while plates are only barcoded on C-segment side, so there is a possibility of low-level barcode exchange (index hopping) across matching wells between plates sequenced on one lane.
8. Troubleshooting
8. Troubleshooting

AB
ProblemPotential Solution(s)
Short peaks (<300bp) are present in the QCRepeat library clean up with lower bead:sample ratio (e.g. 0.8:1).
Faint band/low yieldRepeat PCR II with higher number of cycles
Some wells seem empty or generate unexpectedly low numbers of unique clones after sequencingIf there is a clear geometric pattern, make sure the non-contact liquid dispenser is well calibrated (dispensing expected volume of liquid exactly in the center of the well) using food coloring.
If only the last wells are empty, check if you run out of liquid early during the dispense and increase cell suspension volume.
If only the last wells are empty, check if you run out of liquid early during the dispense and increase cell suspension volume.

9. Data Analysis
9. Data Analysis
See https://github.com/pogorely/TIRTL for guidance on data analysis

10. Appendix I: Single-cell TIRTL-seq
10. Appendix I: Single-cell TIRTL-seq
In certain cases you might be interested in sequencing a very low number of cells or while simultaneously measuring surface expression of markers of interest on sequenced cells. In this case you can perform the protocol after index sorting a single cell/well directly into a 384-well plate pre-loaded with VaporLock and Lysis+RT master mix with the following modifications:
  1. Increase the number of PCR II cycles to 18.
  2. Perform two successive cleanups 0.7:1 Ampure XP:sample ratio to exclude short primer-dimer band.
  3. Sequence at 0.5-1 million 2x150nt reads per plate on any Illumina sequencer.
  4. Do not run combinatorial TIRTL-seq analysis on resulting data: resulting mixcr cloneset usually have just one (or two for double alphas) major clones per well with additional low frequency clonotypes resulting from sequencing errors. Just filtering out low frequency erroneous clones (e.g. <50 reads or <10% readFraction) is sufficient to call paired TCR for each well.