Rt pcr buffer

Following 3 doses (i.d.) of mCherry pDNA or a 1:1 mix of AI and BS pDNA, ILNs and PLNs were collected, and cells were restimulated with PMA (150 ng/ml) and ionomycin (750 ng/ml) for 3 hours in vitro and stained as above. 2.5mi-tetramer (tetr)⁺ CD4⁺ T cells were single-cell sorted (with indexing) using the BD Influx into 96-well plates containing RT-PCR buffer (Qiagen). Single-cell mRNA sequencing of paired TCRα/β and selected T-cell cytokines and transcription factors were achieved by a series of three nested PCR reactions, barcoding and sequencing on MiSeq (Illumina) as described [25 (link)]. For the TCR sequencing reaction, multiple internally nested TCRVα, TCRVβ, TCRCα and Cβ primers were used.

For this experiment, an affected PI HIS mouse with both human and mouse thymi was used. At week 24, when the disease developed, CD3⁺ T cells from skin, liver, spleen, native thymus and pancreatic islets of an affected mouse were sorted using FACS. SP-CD4 and SP-CD8 T cells from grafted human thymus of the same mouse were also sorted. Cells were sorted directly into RT-PCR buffer (Qiagen) using an Influx cell sorter (Becton Dickinson). Index sorting was performed to collect marker information for each single cell. The sequences of expressed TCRα and TCRβ chains from single cells were obtained by a series of three nested PCR reactions with multiple internally nested TCRVα, Vβ, Cα, and Cβ primers as previously described [14 (link)]. Reaction products were barcoded and sequencing was performed on a MiSeq System (Illumina). In addition to TCR sequencing, gene expression level for multiple cytokines and transcription factors that influence T-cell function and define certain T-cell subsets were obtained simultaneously [14 (link)].