GSM2092562: TCR94B.BC8; Homo sapiens; OTHER
Total RNA was isolated from cryofrozen human samples using the TissueLyzer system with Qiazol and steel beads (Qiagen).  RNAseq was performed on total RNA through the Columbia Genome Center, and concentration and RIN score obtained by BioAnalyzer.  For TCR library preparation, mRNA was isolated using magnetic oligo-dT Dynabeads (Life Technologies), according to manufacturer protocol, and final concentration determined by Qubit (Life Technologies).  Total RNA was obtained from 2.5x10^6 PBMCs using the Qiagen RNeasy system according to manufacturer instructions. We utilized the commercially available iRepertoire primer set for nested amplicon rescued multiplex PCR (arm-PCR) of the complementarity-determining region 3 (CDR3) of the human TCR and TCR chains and addition of adaptors for Illumina platform sequencing.  ~400ng of mRNA or total RNA was included for tumor libraries or PBMC libraries respectively.  In our modification of the manufacturer’s protocol, reverse transcription was conducted with a one-step reverse transcription and amplification kit (Qiagen) at 50°C for 40min, 95°C for 15min, seventeen non-exponential thermocycles (94°C/30s, 60°C/2min, 72°C/30s) and ten exponential thermocycles (94°C/30s, 72°C/2min).  PCR1 product was purified using AmpureX-100 magnetic beads (Agencourt).  Secondary amplification of 40% of the resulting product was performed by multiplex PCR (Qiagen), allowing addition of Illumina adapter sequences, with 34 thermocycles (94°C/30s, 55°C/30s, 72°C/30s).  Libraries were purified by agarose gel electrophoresis, cutting between 200-400bp (predicted amplicon size 210-310bp), gel extracted (Qiagen), and sequenced on an Illumina MiSeq, obtaining paired-end reads 220-250nt as described below.
GEO Accession
GSM2092562