Miniseq instrument

DNA extraction and sequencing was performed as previously described [22] (link). A single E. coli colony was grown in Luria-Bertani (LB) broth overnight at 37 ± 2 °C under aerobic conditions. Genomic DNA (gDNA) was extracted using the Wizard Genomics DNA Purification Kit (Promega Corporation, Madison, WI, USA) following the manufacturer's instructions. The quality of gDNA (i.e., purity and integrity) was assessed by a spectrophotometry method using a DeNovix DS-11 spectrophotometer (DeNovix Inc., Wilmington, DE, USA) and 1.0% agarose gel electrophoresis, respectively. Subsequently, the gDNA was quantified using a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA) and appropriately adjusted for paired-end (PE) DNA library preparation using the Nextera XT DNA Library Preparation kit (Illumina, San Diego, CA, USA) following the manufacturer's protocol. Genomic sequencing was performed on an Illumina MiniSeq instrument using a 2 × 150 bp strategy. The raw genomic data of the UTI-U7 isolate was deposited into the Sequence Read Archive (SRA) (accession number SRR21677288) of the National Center for Biotechnology Information (NCBI) linked to BioProject PRJNA883009.

25 mg of frozen liver tissue was lysed to isolate ~25 μg genomic DNA using DNeasy blood & tissue kits (Qiagen). Adaptor oligonucleotides were synthesized by IDT (Supplementary Table 3). Transposon assembly was done by incubating 158ug Tn5 with 1.4 nmol annealed oligo (contains the full-length Illumina forward (i5) adapter, a sample barcode, and UMI)^{40 (link)} at room temperature for 60 min.
For tagmentation, 200 ng of genomic DNA was incubated with 2 μl of assembled transposome at 55° for 7 min, and the product was cleaned up (20 μl) with a Zymo column (Zymo Research, #D4013). Tagmented DNA was used for the 1st PCR using PlatinumTM SuperFi DNA polymerase (Thermo) with i5 primer and gene-specific primers (Supplementary Table 3). Four different libraries were prepared for gDNA from each mouse with different combinations of primers (i5+Locus_F [UDiTaS], i5+Locus_R [UDiTaS], i5+Insert_F [GUIDE-tag] and i5+Insert_R [GUIDE-tag]). The i7 index was added in the 2nd PCR and the PCR product was cleaned up with Ampure XP SPRI beads (Agencourt, 0.9X reaction volume). Completed libraries were quantified by Tapestation and Qubit (Agilent), pooled with equal mole, and sequenced with 150 bp paired-end reads on an Illumina MiniSeq instrument.

mRNA was extracted using the Dynabeads mRNA DIRECT purification kit according to the manufacturer instructions (ThermoFisher, cat# 61012). First strand cDNA was synthesized using oligo dT and the Superscript III (Thermofisher). Second strand was performed using a collection of TRAV/TRBV specific primer (1 cycle with the Phusion from NEB). TCRs were then amplified by PCR (20 cycles with the Phusion from NEB) with a single primer pair binding to the constant region and the adapter linked to the TRAV/TRBV primers added during the reverse transcription. A second round of PCR (25 cycles with the Phusion from NEB) was performed to add the Illumina adapters containing the different indexes. The TCR products were purified with AMPure XP beads (Beckman Coulter), quantified and loaded on the MiniSeq instrument (Illumina) for deep sequencing of the TCRα/TCRβ chain. The TCR sequences were further processed using ad hoc Perl scripts to: (i) pool all TCR sequences coding for the same protein sequence; (ii) filter out all out-frame sequences; (iii) determine the abundance of each distinct TCR sequence. TCR with a single read were not considered for the analysis. This methodology was previously reported in Schmidt et al.^{29 (link)}.

Total RNA from cell culture supernatants was used for NGS. cDNA libraries were constructed using TruSeq Stranded mRNA kit (Illumina), following a modified version of the manufacturer’s protocol that skipped steps necessary for mRNA purification and for rRNA depletion. Sequencing was performed using the paired-end 2 × 150 chemistry on an Illumina MiniSeq instrument. Nucleotide and aa sequence alignments were generated using CLC Genomics Workbench 9.1 (www.clcbio.com). The same software package was used to perform the analysis of next-generation sequencing data, including read mapping, contig assembly, and variant detection. Additional sequencing details including the number of mapped reads, the length of the consensus sequence, % coverage of reference sequence, the number of ambiguous or missing nt at the termini, and the average coverage, are shown in S1 Table. The raw data will be available upon request.

mRNA was isolated using the Dynabeads mRNA DIRECT Purification Kit (Life Technologies) and was amplified using the MessageAmp II aRNA Amplification Kit (Ambion) with the following modifications: IVT was performed at 37 °C for 16 h. First, strand cDNA was synthesized using SuperScript III (Thermo Fisher Scientific) and a collection of TRAV/TRBV-specific primers. TCRs were then amplified by PCR (20 cycles with the Phusion from New England Biolabs) with a single primer pair binding to the constant region and the adapter linked to the TRAV/TRBV primers added during the reverse transcription. A second round of PCR cycle (25 cycles with the Phusion from New England Biolabs) was performed to add the Illumina adapters containing the different indexes. The TCR products were purified with AMPure XP beads (Beckman Coulter), quantified and loaded on the MiniSeq instrument (Illumina) for deep sequencing of the TCRα/TCRβ chain. The TCR sequences were further processed using ad hoc Perl scripts to (1) pool all TCR sequences coding for the same protein sequence; (2) filter out all out-frame sequences; and (3) determine the abundance of each distinct TCR sequence. TCR sequences with a single read were not considered for analysis.

mRNA was extracted using the Dynabeads mRNA DIRECT purification kit according to the manufacturer instructions (ThermoFisher, cat# 61012) and was then amplified using the MessageAmp II aRNA Amplification Kit (Ambion, cat# AM1751) with the following modifications: in vitro transcription was performed at 37 °C for 16 h. First strand cDNA was synthesized using the Superscript III (Thermofisher) and a collection of TRAV/TRBV specific primers. TCRs were then amplified by PCR (20 cycles with the Phusion from NEB) with a single primer pair binding to the constant region and the adapter linked to the TRAV/TRBV primers added during the reverse transcription. A second round of PCR (25 cycles with the Phusion from NEB) was performed to add the Illumina adapters containing the different indexes. The TCR products were purified with AMPure XP beads (Beckman Coulter), quantified and loaded on the MiniSeq instrument (Illumina) for deep sequencing of the TCRα/TCRβ chain. The TCR sequences were further processed using ad hoc Perl scripts to: (i) pool all TCR sequences coding for the same protein sequence; (ii) filter out all out-frame sequences; (iii) determine the abundance of each distinct TCR sequence. TCR with a single read were not considered for the analysis. This methodology was previously reported in Arnaud et al. and Bobisse et al.^{31 (link),44} .

Libraries were prepared using the NEXTFLEX® rapid XP DNA-seq 2.0 kit for Illumina platforms (PerqkinElmer, Waltham, MA, USA). The quantification of the libraries was carried out using the Qubit dsDNA HS assay kit (Thermo Fisher Scientific, Waltham, MA, USA), and the size of the libraries was measured using the Agilent TapeStation 4200 (Agilent Technologies, Santa Clara, CA, USA). High-quality libraries were pooled to achieve equimolar concentrations, 1% PhiX Control v3 Library (Illumina, San Diego, CA, USA) was added, and then next-generation paired-end sequencing (2 × 150 bp) was performed on an Illumina MiniSeq instrument using the 300-cycle MiniSeq Mid Output Reagent Cartridge (Illumina, San Diego, CA, USA).