Mar 17, 2023

Slide-TCR-Seq v3 (IVT) V.2

DOI
https://dx.doi.org/10.17504/protocols.io.n92ldp6w8l5b/v2
  • 1Biophysics Program, Harvard University, Boston, MA 02115, USA;
  • 2Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA;
  • 3Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA;
  • 4Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA
Ruth Raichur
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DOI: https://dx.doi.org/10.17504/protocols.io.n92ldp6w8l5b/v2
Protocol Citation: Sophia Liu, Ruth Raichur, Fei Chen 2023. Slide-TCR-Seq v3 (IVT). protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldp6w8l5b/v2Version created by Ruth Raichur
Manuscript citation:
Liu, Sophia, et al. “Spatial Maps of T Cell Receptors and Transcriptomes Reveal Distinct Immune Niches and Interactions in the Adaptive Immune Response.” Immunity, vol. 55, no. 10, 2022, https://doi.org/10.1016/j.immuni.2022.09.002.


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 16, 2023
Last Modified: March 17, 2023
Protocol Integer ID: 78915
Keywords: T cell receptor, spatial transcriptomics, tertiary lymphoid structures, cancer niches, Slide-TCR-seq, IVT, in vitro transcription, tcr repertoire, specific immune response, tcrs within intact tissue, cell receptor, cell clonotype, tcr, whole transcriptome, sequencing method, cell clonotypes in tissue, cell response, seq v3, transcription, neighboring cell type, transcription in lieu
Abstract
T cells mediate antigen-specific immune responses to disease through the specificity and diversity of their clonotypic T cell receptors (TCRs). Determining the spatial distributions of T cell clonotypes in tissues is essential to understanding T cell behavior, but spatial sequencing methods remain unable to profile the TCR repertoire.

We developed Slide-TCR-seq, a 10-μm-resolution method, to sequence whole transcriptomes and TCRs within intact tissues. Our method yields insights into the spatial relationships between clonality, neighboring cell types, and gene expression that drive T cell responses.

The most recent version of our protocol uses in vitro transcription in lieu of rhPCR amplification, which overcomes the barcode switching introduced by the rhPCR and results in higher mapping rates.
Materials
LIBRARY PREPARATION
  • 1.5 mL Eppendorf LoBind Tubes (Eppendorf, 0030122275)
  • 0.2 mL TempAssure PCR Flex-Free 8-Tube Strips, Attached Individual Optical Caps (USA Scientific, 1402-4700)
  • UltraPure Distilled Water (Invitrogen, 10977015)
  • NxGen RNase Inhibitor (Lucigen, F83923-1)
  • Maxima H minus Reverse Transcriptase + Maxima 5X RT Buffer (Thermo Scientific, EP0752)
  • Deoxynucleotide (dNTP) solution mix (New England BioLabs, N0447L)
  • AmPure XP (SPRI beads) (Beckman Coulter, A63881)
  • SPRIselect (SPRI beads) (Beckman Coulter, B23319)
  • Qubit dsDNA HS Assay Kit (Thermofisher, Q32851)
  • Bioanalyzer High Sensitive DNA kit (Agilent, 5067-4626)
  • HiScribe™ T7 Quick High Yield RNA Synthesis Kit (New England BioLabs, E2050S)
  • 2x KAPA Hifi Hotstart Readymix (Roche, KK2602)

OLIGONUCLEOTIDE SEQUENCES
AB
Truseq-P5 HybridAATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT
T7 PCR primerTCTAGATAATACGACTCACTATAGGG
Human T7-TCRV primer mix See table 2
Mouse T7-TCRV primer mix See table 3
Table 1: Oligonucleotide sequences
AB
NameSequence
TRAV1 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGGTCGTTTTTCTTCATTCCTTAGTC
TRAV2 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGACGATACAACATGACCTATGAACGG
TRAV3.1 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTTTGAAGCTGAATTTAACAAGAGCC
TRAV4.1 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTCCCTGTTTATCCCTGCCGAC
TRAV5.1 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGAAACAAGACCAAAGACTCACTGTTC
TRAV6 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGAAGACTGAAGGTCACCTTTGATACC
TRAV7 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGACTAAATGCTACATTACTGAAGAATGG
TRAV8 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCATCAACGGTTTTGAGGCTGAATTTAA
TRAV9 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGGAAACCACTTCTTTCCACTTGGAGAA
TRAV10 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGTACAGCAACTCTGGATGCAGACAC
TRAV12 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGGAAGATGGAAGGTTTACAGCACA
TRAV13.1 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACATTCGTTCAAATGTGGGCGAA
TRAV13.2 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGCAAGGCCAAAGAGTCACCGT
TRAV14 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCCAGAAGGCAAGAAAATCCGCCA
TRAV16 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCTGACCTTAACAAAGGCGAGACA
TRAV17 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGTTAAGAGTCACGCTTGACACTTCCA
TRAV18 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCAGAGGTTTTCAGGCCAGTCCT
TRAV19 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCCACCAGTTCCTTCAACTTCACC
TRAV20 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCCACATTAACAAAGAAGGAAAGCT
TRAV21 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCCTCGCTGGATAAATCATCAGGA
TRAV22 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGACGACTGTCGCTACGGAACGCTA
TRAV23 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGCACAATCTCCTTCAATAAAAGTGCCA
TRAV24 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGACGAATAAGTGCCACTCTTAATACCA
TRAV25 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGGTTTGGAGAAGCAAAAAAGAACAGCT
TRAV26.1 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAGAAGACAGAAAGTCCAGCACCT
TRAV26.2 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGATCGCTGAAGACAGAAAGTCCAGT
TRAV27 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGACTAACCTTTCAGTTTGGTGATGCAA
TRAV29 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTTAAACAAAAGTGCCAAGCACCTC
TRAV30 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGAATATCTGCTTCATTTAATGAAAAAAAGC
TRAV34 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCAAGTTGGATGAGAAAAAGCAGCA
TRAV35 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTCAGTTTGGTATAACCAGAAAGGA
TRAV36 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGAAGACTAAGTAGCATATTAGATAAG
TRAV38 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTGTGAACTTCCAGAAAGCAGCCA
TRAV39 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCTCACTTGATACCAAAGCCCGT
TRAV40 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGGCGGAAATATTAAAGACAAAAACTC
TRAV41 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGGATTAATTGCCACAATAAACATACAGG
TRBV2 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCCTGATGGATCAAATTTCACTCTG
TRBV3-1 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCTCACCTAAATCTCCAGACAAAGCT
TRBV4 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCTGAATGCCCCAACAGCTCTC
TRBVS-48 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTCTGAGCTGAATGTGAACGCCT
TRBVS-1 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGCGATTCTCAGGGCGCCAGTTCTCT
TRBV6-1 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGGCTACAATGTCTCCAGATTAAACAA
TRBV6-23 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCCTGATGGCTACAATGTCTCCAGA
TRBV6-4 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGGTGTCTCCAGAGCAAACACAGATGATT
TRBV6-56 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGGTCTCCAGATCAACCACAGAGGAT
TRBV6-8 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGGTCTCTAGATTAAACACAGAGGATTTC
TRBV6-9 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGCTACAATGTATCCAGATCAAACA
TRBV7-2 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCGCTTCTCTGCAGAGAGGACTGG
TRBV7-3 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGCGGTTCTTTGCAGTCAGGCCTGA
TRBV7-8 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCAGTGATCGCTTCTTTGCAGAAA
TRBV?-46 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCTCCACTCTGAMGATCCAGCGCA
TRBV7-7 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCAGAGAGGCCTGAGGGATCCAT
TRBV7-9 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTGCAGAGAGGCCTAAGGGATCT
TRBV9 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTCCGCACAACAGTTCCCTGACTT
TRBV10-13 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAGATGGCTAYAGTGTCTCTAGATCAAA
TRBV10-2 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGGTTGTCTCCAGATCCAAGACAGAGAA
TRBV11 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCAGAGAGGCTCAAAGGAGTAGACT
TRBV12-34 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCTAAGATGCCTAATGCATCATTCTC
TRBV12-5 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTCAGCAGAGATGCCTGATGCAACT
TRBV13 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCTCAGCTCAACAGTTCAGTGACTA
TRBV14 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCTGAAAGGACTGGAGGGACGTAT
TRBV15 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGGATAACTTCCAATCCAGGAGGCCG
TRBV16 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCTAAGTGCCTCCCAAATTCACCC
TRBV18 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGAACGATTTTCTGCTGAATTTCCCA
TRBV19 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGTACAGCGTCTCTCGGGAGAAGA
TRBV20-1 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACAAGTTTCTCATCAACCATGCAA
TRBV24-1 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGTGGATACAGTGTCTCTCGACAGGC
TRBV25-1 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAACAGTCTCCAGAATAAGGACGGA
TRBV27-1 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGTACAAAGTCTCTCGAAAAGAGAAGAGGA
TRBV28 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGGGTACAGTGTCTCTAGAGAGA
TRBV29 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGGTTTCCCATCAGCCGCCCAAACCTA
TRBV30 TCTAGATAATACGACTCACTATAGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGCAGACCCCAGGACCGGCAGTTCAT
Table 2: Human T7-UPS2-TCRV oligo sequences

AB
mAV01AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCCGCTCGAATGGGTACAGTTACCTGA
mAV02AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCGGAAGCTCAGCACTCTGAACCTGA
mAV03AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACACTCTCTCTGAACCTCACAGCTGCCCAA
mAV041AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCGCTACAGCACCCTGCACATCAC
mAV042AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACTTCTAAGGAGAGCTACAGCACCCTGCAA
mAV043AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACTTCTAAGGAGAGCTACAGCACCCCGCAA
mAV044AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACTTCTAAGGAGCTCTACAGCACCCTGCAA
mAV051AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACTTACAGCCACTCAGCCTGGAGACTA
mAV052AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCACAGACACCCAGCCTGGAGACA
mAV061AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCCTTCCACTTGCAGAAAGCCTCAGTA
mAV062AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACTCCTTCCACTTACAGAAAGCCTCAGTGCT
mAV063AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACGGAAGCAGCAGAGGTTTTGAAGCTACAC
mAV071AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCAGCTCACCTCAATAAGGCCAGCCTGA
mAV072AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACGAGACTCCCAGCCCAGTGACTCA
mAV073AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCAGAGAGTCGCAACCCAGTGACTCA
mAV074AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACTCAATAGAGCCAGCCTGCATGTTTCA
mAV075AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACGTGTCCATCTTCTCTGATGGTGAAAAGGT
mAV081AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCACCCTTGACACCTCCAGCCT
mAV082AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCAGTGGAAGACTCAGAGCCACCCTTA
mAV091AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACGAAAGCCTCCGTGCACTGGAGCGT
mAV092AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACGCTTCGAGGCTGAGTTCAGCAAGAC
mAV093AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGTAACTCTTCCTTCCACCTGCGGAAAT
mAV10AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACATCACAGCCACACAGCCTGAAGAC
mAV11AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCACAGCACGCTGCACATCACAGA
mAV12AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCAGCTCCTTCCATCTGCAGAAGTCCA
mAV131AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACGCTCTTTGCACATTTCCTCCTCCCAGAA
mAV132AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACGCTCTTTGACTATATCCTCCTCCCAGACCT
mAV141AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGACTCTCAGCCTGGAGACTCAGCCT
mAV142AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGGAAGATGGACGATTCACAATCTTCTTCAC
mAV151AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCCGCTATTCTGTAGTCTTCCAGAAATCACA
mAV152AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCCATCAGCCTTGTCATTTCAGCCTCACT
mAV16AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCAAAAAGTTCCATCGGACTCATCAC
mAV17AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCTTTCAACCTGAAGAAATCCCCAGCCCATA
mAV19AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCTTCTCACTGCACATCACAGCCTCCCT
mAV21AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACTGGCTATTGCCTCTGACAGAAAGTCAC
mBV01AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCTGATACGGAGCTGAGGCTGCAAGA
mBV02AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACTCAGATCACAGCTCTAAAGCCTGATGACC
mBV03AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCCAACCCACAGCACTGGAGGACA
mBV04AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCGCTTCTCACCTCAGTCTTCAGATAAC
mBV05AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACTGCCCAGACAGCTCCAAGCTACA
mBV12AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCCCAGCAGATTCTCAGTCCAACAGTC
mBV131AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACGCCACCAGAACAACGCAAGAAGC
mBV132AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACACAAGGCCTCCAGACCAAGCCAAT
mBV14AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACGCCTAAAGGAACTAACTCCACTCTCAAGAC
mBV15AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACTGAAGATTCAACCTACAGAACCCAAGGACA
mBV16AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACTGAAGATCCAGAGCACGCAACCCCT
mBV17AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACTCTCTACATTGGCTCTGCAGGCCTAGT
mBV19AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACTCACTGTGACATCTGCCCAGAAGAT
mBV20AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACTTCCCATCAGTCATCCCAACTTATCCTA
mBV23AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACTCTGCAGCCTGGGAATCAGAACGA
mBV24AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACGCATCCTGGAAATCCTATCCTCTGAAGAC
mBV25AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCCCAATCTCATCCTTCATCTTGGAAATGCT
mBV26AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACTGCAGCCTAGAAATTCAGTCCTCTGAGA
mBV29AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACGGGAGCATTTCTCCCTGATTCTGGATTA
mBV30AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACGCCAAACCTAACATTCTCAACGTTGACAGA
mBV31AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACACGGAGAAGCTGCTTCTCAGCCACA
Table 3: Mouse T7-UPS2-TCRV oligo sequences

PCR to add T7 to cDNA libraries
This protocol amplifies TCRs from unfragmented, full-length cDNA from Slide-Seq.
Prepare two 10-nanogram* dilutions of all samples into 12.25 μL of ultrapure water for amplifying TCR alpha and beta sequences in separate reactions.
*We have successfully tested down to 2 ng for low-concentration samples.
Prepare the primer extension PCR master mix using KAPA Hifi Hotstart Readymix 2X and T7-TCRV primer pool. Gently mix by pipetting and run the PCR program below.

Note: TRAV and TRBV primers are pooled separately and are treated as individual reactions for each sample.

Primer extension PCR mix per sample:
ABC
Volume (μL)ReagentFinal concentration
12.5KAPA Hifi Hotstart Readymix 2X1 X
0.25100 μM T7-TCRV primer pool1 μM
4-10 ngcDNA
up to 25 μL Ultrapure water
Final volume 25 μL

Primer extension PCR protocol:
ABC
CyclesTempTime
595 °C5 minutes
65 °C for human primer pool/70 °C for mouse primer pool30 seconds
72 °C3 minutes
14 °Chold
Safe stopping point, store at 4 °C
Add 25 μL of water to bring the reaction to 50 μL. Perform a PCR clean-up following the manufacturer's instructions using SPRIselect or AMPure XP beads at 0.6X (30 μL of SPRI beads to 50 μLPCR reaction volume). Elute in 9 μL of water.
Prepare the T7/Truseq PCR master mix with KAPA Hifi Hotstart Readymix 2X. Add 16 μL of master mix to 9 μL of the sample. Gently mix by pipetting and run the PCR program below.

T7/Truseq PCR mix per sample:
ABC
Volume (uL)ReagentFinal concentration
12.5KAPA Hifi Hotstart Readymix 2X1 X
0.5100uM Truseq-P5 Hybrid primer2 μM
0.5100uM T7 PCR primer2 μM
9Sample
2.5Ultrapure water (up to 25)
Final volume 25 μL

T7/Truseq PCR protocol:
ABC
CyclesTempTime
198 °C2 minutes
1098 °C1 minute
60 °C30 seconds
72 °C3 minutes
172 °C5 minutes
4 °Chold
Safe stopping point, store at 4 °C
Add 25 μL of water to bring the reaction to 50 μL. Perform a PCR clean-up following the manufacturer's instructions using SPRIselect or AMPure XP beads at 0.6X (30 μL of SPRI beads to 50 μLPCR reaction volume). Elute in 8 μL of water.
IVT amplification
Follow the manufacturer's instructions on the HiScribe RNA synthesis kit using 8 μL of the sample eluted in the previous step. Incubate reaction for 2 hours at 37 °C.

HiScribe RNA Synthesis mix per sample:
ABC
Volume (uL)ReagentFinal concentration
10NTP Buffer Mix10 mM each NTP
2T7 RNA Polymerase Mix
8Sample
Final volume 20 μL

Use RNAse away to clean all surfaces and pipettors.
Add 30 μL of water to bring the reaction to 50 μL. Perform a PCR clean-up using SPRIselect or AMPure XP beads at 0.6X (30 μL of SPRI beads to 50 μL PCR reaction volume), following the steps below:
For a 50 μL reaction, add 30 uL of SPRI beads.
Incubate for 5 minutes at RT.
Incubate for 2 minutes on a magnet until the solution turns clear.
Discard supernatant.
Wash on a magnet for 30 sec with 200 μL of freshly made 80% EtOH.
Repeat wash.
Discard supernatant.
Spin down briefly on a table spinner.
Remove all EtOH with a 20 μL pipette.
Elute with 20 μL of H2O.
Use a NanoDrop on the RNA setting to measure RNA concentration.
RT
Add 180 μL of the following RT mix to 20 μL of the RNA sample. Incubate reaction for 2 hours at 42 °C.

Reverse Transcription Mix per sample:
ABC
Volume (uL)ReagentFinal concentration
40Maxima 5X RT buffer1 X
2010 mM dNTPs1 mM
5RNAse inhibitor
2100 μM Truseq-P5 Hybrid primer1 μM
10Maxima H-RTase
20Template RNA (sample)1 pg - 5 ug
103Ultrapure water (up to 200)
Final volume 200 μL

Safe stopping point, store at 4 °C
Perform a PCR clean-up following the manufacturer's instructions using SPRIselect or AMPure XP beads at 0.6X (120 μL of SPRI beads to 200 μL RT reaction volume). Elute in 20 μL of water. Record concentrations using a NanoDrop on the ssDNA setting and save all samples.

Index PCR
Prepare the PCR master mix with KAPA Hifi Hotstart Readymix 2X, P5-Truseq PCR primer, and Nextera PCR primer index. Gently mix by pipetting and divide the total volume of each sample into 4 PCR tubes each containing 50 μL (25%) of the total.

Note: Each sample must use a different i7 index if you intend to pool samples for multiplexed sequencing. We do not recommend dual-indexing of samples.

Index PCR mix per sample:
ABC
Volume (uL)ReagentFinal Concentration
100KAPA Hifi Hotstart Readymix 2X1 X
4100 μM Truseq-P5 Hybrid PCR primer2 μM
4100 μM Nextera PCR primer (i7)2 μM
100 ngSample
up to 200ultrapure water
Final volume 200 μL

Index PCR protocol:
ABC
CyclesTempTime
198 °C2 minutes
1098 °C1 minute
67 °C20 seconds
72 °C3 minutes
172 °C5 minutes
4 °Chold

Safe stopping point, store at 4 °C
Recombine the samples that were split into 4 parts in the previous step and perform PCR clean-up following the manufacturer's instructions using SPRIselect or AMPure XP beads at 0.6X (120 μL of SPRI beads to 200 μL PCR reaction volume). Elute in 10 μL of water.

To quantify the TCR libraries, use the Qubit dsDNA high-sensitivity kit and BioAnalyzer High-SensitivityThe expected DNA kit following the manufacturer protocols.
BioAnalyzer trace of a TRB library. The expected library length is around 1100bp.
BioAnalyzer trace of a TRA library. The expected library length is around 1300bp.

Sequencing
TRA libraries are best sequenced on a Nanopore. TRB libraries can be sequenced on a Nanopore or MiSeq.
For best results, it's generally advised to sequence each sample to a depth of 1-2 million reads.

MiSeq read structure is as follows:
Read 1: 42 bp
Index 1: 8 bp
Read 2: 270 bp
Index 2: 0 bp