GSM3466800: P01_pp65mLCL_all_beta; Homo sapiens; OTHER
RNA extraction was performed on bulk cells (either unsorted or CD8-enriched using MACS) with the Qiagen RNeasy kit. 1 μg RNA per sample was reversely transcribed to cDNA with the QuantiTect Reverse Transcription Kit (Qiagen) using a primer designed to target both the Cβ1 and Cβ2 regions of the TCR RNA. cDNA was amplified in two subsequent PCRs using Pfu DNA polymerase (Thermo Scientific). The first PCR was a multiplex PCR comprising 42 distinct forward primers that bind to the Vβ region and cover all possible human TCR Vβ segments, and a reverse primer that anneals to the Cβ1 and Cβ2 regions. Forward and reverse primers carried sequences complementary to Illumina Read 2 and Read 1 primer sequences. In the second PCR, index primers (NEBNext Multiplex Oligos for Illumina; New England BioLabs) were used to attach barcodes and the i5 and i7 adapters for binding to the Illumina flow cell (Supplementary Fig. 2). After each PCR step, the PCR product was purified using Agencourt AMPure XP magnetic beads (Beckman Coulter). The correct length and amount of the PCR products for sequencing was confirmed using the Agilent DNA 1000 Kit and the Bioanalyzer 2100 (Agilent). To enhance the nucleotide diversity of TCR reads facilitating cluster recognition and avoiding artifacts in q-score calibration and base calling, three different forms of the reverse primer, with 0, 1 or 2 arbitrary nucleotides between the Cβ-binding sequence and the Illumina Read 1 sequence were used. All primers were used in equimolar amounts (a total of 10μM; for primer sequences see Supplementary Table 1). The first PCR consisted of only 10 amplification cycles to minimize PCR amplification bias.
GEO Accession
GSM3466800