Sequencing primer

Aliquots of 2 μl amplified cDNA from the single-cell expression library construction workflow were used for TCR library construction according to 10x genomics manufacturer’s manual. Quality of amplified cDNA and constructed libraries were confirmed by BioAnalyzer (Agilent, High Sensitivity DNA Kit). Library sequencing was performed by NextSeq 500 (Illumina). Sequencing depth for V(D)J enriched libraries were at least 5000 read pairs per cell. Standard Illumina sequencing primers were used for both sequencing and index reads following 10x manufacturer’s protocol.

The procedure of high throughput Illumina sequencing was carried out as previously described^{46 (link)}. Resulting sequences were analyzed using “Galaxy Bioinformatics”, a free software at https://usegalaxy.org/ and “FastQC” version 0.11.5, a free software at www.bioinformatics.babraham.ac.uk^{49 (link)–51 (link)}. Samples from rounds 1, 6, 8 and 10 were prepared for Illumina sequencing. Each selected round’s ssDNA was barcode-tagged and used as a template to be amplified by PCR using Illumina sequencing primers (Supplementary Table S1). Twenty-five PCR cycles have been applied using KOD hot start DNA polymerase and buffer (Sigma-Aldrich, CA). All PCR products were sent for sequencing at the University of Wisconsin Biotechnology Center (UWBC, WI).
Total number of the reads by the Illumina sequencing were ~ 16 million reads; however, 5% of the total reads were neglected due to their improper length or quality. The rest of the reads were tracked according to their barcodes. After analyzing the results, the highly-yielded aptamers were tested for the free energy change (ΔG[kcal.mole⁻¹]) of the three dimensional structure using the OligoAnalyzer 3.1 Tool at Integrated DNA technologies website (www.idtdna.com) and the most reliable ones have been chosen to be tested by the next step.

RNA-seq experiments were performed as previously described [12 (link)] with modifications. Briefly, total RNA from mouse cortical tissues or cultured cells was extracted using TRIzol reagents (Life Technologies) following the manufacturer’s instructions. Total mRNA was prepared from 1 μg total RNA using poly(A) mRNA magnetic isolation reagents (NEB) and fragmented at 94 °C for 15 min. RNA was then reverse-transcribed into cDNA with random primers. After end repairing and A-tailing, cDNA was ligated with adapters and amplified by PCR with Illumina sequencing primers. All RNA-seq experiments were performed with biological replicates. RNA-seq libraries were sequenced on a HiSeq X Ten platform.

GBS data was generated at the Institute of Biotechnology, Cornell University as described previously^{30 (link)}. Briefly, DNA samples were digested with the restriction enzyme PstI followed by ligation of adapters, that consisted of Illumina sequencing primers and barcode adapters, to the DNA fragment ends. After ligation, 95 samples were combined into a pool and PCR amplification was performed to create a GBS library and sequenced on Illumina HiSeq 2000.

The total RNA extraction kit (TIANGEN) was used to extract total RNA from lymphocytes in strict accordance with the operation steps in the instructions. We obtained mononuclear cells from 2ml peripheral blood using the TRIzol reagent according to the manufacturer's protocol. The concentrations of RNA were evaluated using a NanoDrop ND‐2000 spectrophotometer (Thermo Scientific). A total of 200ng RNA was converted into cDNA by reverse transcription using a Transciptor First Strand cDNA Synthesis Kit (Roche Applied Science, Penzberg, Germany) according to the manufacturer's protocol on a T100TM Thermal Cycler (Bio‐Rad Inc).²⁴ Quality inspection was carried out with Agilent2200, and cDNA was stored at −80°C.
Then, the libraries were constructed following the protocol from Liang et al.²⁵ For TCRβ CDR3 library preparation, two‐round nested amplicon arm‐PCR was performed with a Multiplex PCR Assay Kit Ver.2 (TaKaRa) using specific primers against each variable and constant gene. PCR products were purified by agarose gel electrophoresis, amplified using Illumina sequencing primers with different sample barcodes and subjected to high‐throughput sequencing using the Illumina HiSeq X Ten PE150 platform.

E. coli W3110 frozen cells containing the BH plasmid library were diluted 1:1000 and recovered in 10 ml of MH supplemented with 75 μg/ml carbenicillin for 2 hours. The culture was then back diluted to OD 0.05 and three triplicate 5 ml cultures were set up in MH supplemented with 75 μg/ml carbenicillin. Triplicate reactions included: Uninduced (0 μM IPTG), and induced (100 μM IPTG). All triplicate cultures were then grown for 4 hours at 37°C. Plasmids from each triplicate culture were then miniprepped and Illumina sequencing primers were used to produce an amplicon via PCR (Table S3). Amplicons were sent to Genewiz for next generation sequencing by Illumina MiSeq technology with 30% added Phi-X DNA.

SLAY procedures have been detailed previously (17 (link), 35 (link)). Briefly, E. coli 25922 frozen cells containing the BH plasmid library were diluted 1:1000 and recovered in 10 ml of HS supplemented with 75 μg/ml carbenicillin for 2 hours. The culture was then back diluted to OD 0.05 and three triplicate 5 ml cultures were set up in HS supplemented with 75 μg/ml carbenicillin. Triplicate reactions included: Uninduced (0 μM IPTG), and induced (100 μM IPTG). All triplicate cultures were then grown for 4 hours at 37°C. Plasmids from each triplicate culture were miniprepped and Illumina sequencing primers were used to produce an amplicon via PCR (Table S6). Amplicons were sent to Genewiz for next generation sequencing by Illumina MiSeq technology with 30% added Phi-X DNA.