Mar 16, 2023
Version 2
Slide-TCR-Seq v3 (IVT) V.2
- Sophia Liu1,2,3,
- Ruth Raichur3,
- Fei Chen3,4
- 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
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 16, 2023
Protocol Integer ID: 78915
Keywords: T cell receptor, spatial transcriptomics, tertiary lymphoid structures, cancer niches, Slide-TCR-seq, IVT, in vitro transcription
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
| A | B | |
| Truseq-P5 Hybrid | AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT | |
| T7 PCR primer | TCTAGATAATACGACTCACTATAGGG | |
| Human T7-TCRV primer mix | See table 2 | |
| Mouse T7-TCRV primer mix | See table 3 | |
Table 1: Oligonucleotide sequences
| A | B | |
| Name | Sequence | |
| 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
| A | B | |
| mAV01 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCCGCTCGAATGGGTACAGTTACCTGA | |
| mAV02 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCGGAAGCTCAGCACTCTGAACCTGA | |
| mAV03 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACACTCTCTCTGAACCTCACAGCTGCCCAA | |
| mAV041 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCGCTACAGCACCCTGCACATCAC | |
| mAV042 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACTTCTAAGGAGAGCTACAGCACCCTGCAA | |
| mAV043 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACTTCTAAGGAGAGCTACAGCACCCCGCAA | |
| mAV044 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACTTCTAAGGAGCTCTACAGCACCCTGCAA | |
| mAV051 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACTTACAGCCACTCAGCCTGGAGACTA | |
| mAV052 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCACAGACACCCAGCCTGGAGACA | |
| mAV061 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCCTTCCACTTGCAGAAAGCCTCAGTA | |
| mAV062 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACTCCTTCCACTTACAGAAAGCCTCAGTGCT | |
| mAV063 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACGGAAGCAGCAGAGGTTTTGAAGCTACAC | |
| mAV071 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCAGCTCACCTCAATAAGGCCAGCCTGA | |
| mAV072 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACGAGACTCCCAGCCCAGTGACTCA | |
| mAV073 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCAGAGAGTCGCAACCCAGTGACTCA | |
| mAV074 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACTCAATAGAGCCAGCCTGCATGTTTCA | |
| mAV075 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACGTGTCCATCTTCTCTGATGGTGAAAAGGT | |
| mAV081 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCACCCTTGACACCTCCAGCCT | |
| mAV082 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCAGTGGAAGACTCAGAGCCACCCTTA | |
| mAV091 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACGAAAGCCTCCGTGCACTGGAGCGT | |
| mAV092 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACGCTTCGAGGCTGAGTTCAGCAAGAC | |
| mAV093 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGTAACTCTTCCTTCCACCTGCGGAAAT | |
| mAV10 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACATCACAGCCACACAGCCTGAAGAC | |
| mAV11 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCACAGCACGCTGCACATCACAGA | |
| mAV12 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCAGCTCCTTCCATCTGCAGAAGTCCA | |
| mAV131 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACGCTCTTTGCACATTTCCTCCTCCCAGAA | |
| mAV132 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACGCTCTTTGACTATATCCTCCTCCCAGACCT | |
| mAV141 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGACTCTCAGCCTGGAGACTCAGCCT | |
| mAV142 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGGAAGATGGACGATTCACAATCTTCTTCAC | |
| mAV151 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCCGCTATTCTGTAGTCTTCCAGAAATCACA | |
| mAV152 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCCATCAGCCTTGTCATTTCAGCCTCACT | |
| mAV16 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACAGCCAAAAAGTTCCATCGGACTCATCAC | |
| mAV17 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCTTTCAACCTGAAGAAATCCCCAGCCCATA | |
| mAV19 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCTTCTCACTGCACATCACAGCCTCCCT | |
| mAV21 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACTGGCTATTGCCTCTGACAGAAAGTCAC | |
| mBV01 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCTGATACGGAGCTGAGGCTGCAAGA | |
| mBV02 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACTCAGATCACAGCTCTAAAGCCTGATGACC | |
| mBV03 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCCAACCCACAGCACTGGAGGACA | |
| mBV04 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCGCTTCTCACCTCAGTCTTCAGATAAC | |
| mBV05 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACTGCCCAGACAGCTCCAAGCTACA | |
| mBV12 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCCCAGCAGATTCTCAGTCCAACAGTC | |
| mBV131 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACGCCACCAGAACAACGCAAGAAGC | |
| mBV132 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACACAAGGCCTCCAGACCAAGCCAAT | |
| mBV14 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACGCCTAAAGGAACTAACTCCACTCTCAAGAC | |
| mBV15 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACTGAAGATTCAACCTACAGAACCCAAGGACA | |
| mBV16 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACTGAAGATCCAGAGCACGCAACCCCT | |
| mBV17 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACTCTCTACATTGGCTCTGCAGGCCTAGT | |
| mBV19 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACTCACTGTGACATCTGCCCAGAAGAT | |
| mBV20 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACTTCCCATCAGTCATCCCAACTTATCCTA | |
| mBV23 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACTCTGCAGCCTGGGAATCAGAACGA | |
| mBV24 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACGCATCCTGGAAATCCTATCCTCTGAAGAC | |
| mBV25 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACCCCAATCTCATCCTTCATCTTGGAAATGCT | |
| mBV26 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACTGCAGCCTAGAAATTCAGTCCTCTGAGA | |
| mBV29 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACGGGAGCATTTCTCCCTGATTCTGGATTA | |
| mBV30 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACGCCAAACCTAACATTCTCAACGTTGACAGA | |
| mBV31 | AGATAATACGACTCACTATAGGGTCGTGGGTCGTGGGCTCGGAGATGTGTATAAGAGACACGGAGAAGCTGCTTCTCAGCCACA |
Table 3: Mouse T7-UPS2-TCRV oligo sequences
PCR to add T7 to cDNA libraries
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:
| A | B | C | |
| Volume (μL) | Reagent | Final concentration | |
| 12.5 | KAPA Hifi Hotstart Readymix 2X | 1 X | |
| 0.25 | 100 μM T7-TCRV primer pool | 1 μM | |
| 4-10 ng | cDNA | ||
| up to 25 μL | Ultrapure water |
Final volume 25 μL
Primer extension PCR protocol:
| A | B | C | |
| Cycles | Temp | Time | |
| 5 | 95 °C | 5 minutes | |
| 65 °C for human primer pool/70 °C for mouse primer pool | 30 seconds | ||
| 72 °C | 3 minutes | ||
| 1 | 4 °C | hold |
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:
| A | B | C | |
| Volume (uL) | Reagent | Final concentration | |
| 12.5 | KAPA Hifi Hotstart Readymix 2X | 1 X | |
| 0.5 | 100uM Truseq-P5 Hybrid primer | 2 μM | |
| 0.5 | 100uM T7 PCR primer | 2 μM | |
| 9 | Sample | ||
| 2.5 | Ultrapure water (up to 25) |
Final volume 25 μL
T7/Truseq PCR protocol:
| A | B | C | |
| Cycles | Temp | Time | |
| 1 | 98 °C | 2 minutes | |
| 10 | 98 °C | 1 minute | |
| 60 °C | 30 seconds | ||
| 72 °C | 3 minutes | ||
| 1 | 72 °C | 5 minutes | |
| 4 °C | hold |
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
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:
| A | B | C | |
| Volume (uL) | Reagent | Final concentration | |
| 10 | NTP Buffer Mix | 10 mM each NTP | |
| 2 | T7 RNA Polymerase Mix | ||
| 8 | Sample |
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
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:
| A | B | C | |
| Volume (uL) | Reagent | Final concentration | |
| 40 | Maxima 5X RT buffer | 1 X | |
| 20 | 10 mM dNTPs | 1 mM | |
| 5 | RNAse inhibitor | ||
| 2 | 100 μM Truseq-P5 Hybrid primer | 1 μM | |
| 10 | Maxima H-RTase | ||
| 20 | Template RNA (sample) | 1 pg - 5 ug | |
| 103 | Ultrapure 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
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:
| A | B | C | |
| Volume (uL) | Reagent | Final Concentration | |
| 100 | KAPA Hifi Hotstart Readymix 2X | 1 X | |
| 4 | 100 μM Truseq-P5 Hybrid PCR primer | 2 μM | |
| 4 | 100 μM Nextera PCR primer (i7) | 2 μM | |
| 100 ng | Sample | ||
| up to 200 | ultrapure water |
Final volume 200 μL
Index PCR protocol:
| A | B | C | |
| Cycles | Temp | Time | |
| 1 | 98 °C | 2 minutes | |
| 10 | 98 °C | 1 minute | |
| 67 °C | 20 seconds | ||
| 72 °C | 3 minutes | ||
| 1 | 72 °C | 5 minutes | |
| 4 °C | hold |
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
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