GSM6647585: ALD1_190_liver_bulkTCR_alpha; Homo sapiens; OTHER
Qiagen RNAeasy micro kit miLaboratory Human TCR profiling kit. Liver biopsies were obtained in RNAlater and transferred into 2ml tubes containing 600µl RLT+ buffer + 1% -Mercaptoethanol. Tissue was disrupted using the Qiagen tissue lyser. PBMCs were isolated from paired blood samples by density gradient centrifugation (Biocoll; Biochrom, Berlin, Germany) and CD4+ T cells were enriched using CD4 Microbeads (Miltenyi Biotec). RNA was isolated from liver samples and CD4+ T cells from PBMCs using the RNeasy Micro Kit (Qiagen). TCR libraries were prepared using human TCR RNA multiplex kit (MiLaboratories), according to the manufacturer's protocol using 24 PCR cycles (liver) and 18 PCR cycles (blood) for the 1st PCR, and 14 PCR cycles (liver) and 16 PCR cycles (blood) for the 2nd PCR amplification. Libraries were sequenced on an Illumina NovaSeq6000 machine, SP v1.5 flow cells and 300 cycles. Data of both TCRα and β chains were obtained.