GSM3270828: Heart_MI_1; Mus musculus; OTHER
mice were sacrificed by cervical dislocation, whole-body perfusion with PBS was conducted, and organs were extracted for ex vivo analyses. Lymph nodes cells were obtained after grinding the tissue against a 30um filter mesh in Hank's balanced salt solution. A cardiac cell suspension was obtained after enzimatic digestion with Collagenase-II. CD4+ TCRbeta+ single cells were FACSed (FACS Aria-II, purity > 98%) into RLT lysis buffer (Qiagen,  Hilden, Germany) and stored at -80oC. RNA purification was performed using the Qiagen microKit plus. RNA quality was checked using the Agilent 2100 Bioanalyzer. TCRb-sequencing was outsourced to the specialized company iRepertoire. For the construction of mouse TCR beta chain libraries, iRepertoire used amplicon rescued multiplex PCR (arm-PCR) to amplify each of the 19 submitted RNA samples. The cDNA synthesis protocol involved initial first-round RT-PCR using high concentrations of gene-specific primers (iRepertoire's MTBIvc primers), followed by Beckman Coulter's SPRIselect bead-cleanup procedure, and PCR2 using universal primers to amplify the cDNA exponentially. Each constructed library was measured for concentration and quality using Thermoscientific's Nanodrop 1000, then pooled using 200 ng per constructed library. This pool was QC'ed using a bioanalyzer, KAPA qPCR analysis, and Qubit quantitation. The QC'ed pool was then sequenced according to Illumina's HiSeq sequencing guidelines using a 300-cycle reagent kit. Libraries were pooled and sequenced with the Illumina HiSeq. The output of the mouse TRB sequence covers within the third framework region through the CDR3 and the beginning of the constant region.
GEO Accession
GSM3270828