Illumina HiSeq 2500 sequencing; Somatic Gain-of-Function mTOR mutation in Clonally Expanded T-lymphocytes Associated with Chronic Graft-Versus-Host Disease
Somatic Gain-of-Function mTOR mutation in Clonally Expanded T-lymphocytes Associated with Chronic Graft-Versus-Host Disease
CD4+ T cells from two time points of the index patient were enriched using CD4 microbeads (Miltenyi Biotec). Single cells were partitioned using a Chromium Controller (10x Genomics) and scRNA-seq and TCRαβ-libraries were prepared using Chromium Single Cell 5' Library & Gel Bead Kit (10x Genomics), as per manufacturer's instructions (CG000086 Rev D). ). In brief, 17,000 cells from each sample, suspended in 0.04% BSA in PBS were loaded on the Chromium Single Cell A Chip.  During the run, single-cell barcoded cDNA is generated in nanodroplet partitions. The droplets are subsequently reversed, and the remaining steps are performed in bulk.  Full length cDNA was amplified using 14 cycles of PCR (Veriti, Applied Biosystems). TCR cDNA was further amplified in a hemi-nested PCR reaction using Chromium Single Cell Human T Cell V(D)J Enrichment Kit (10x Genomics).  Finally, the total cDNA and the TCR-enriched cDNA was subjected to fragmentation, end repair and A-tailing, adaptor ligation, and sample index PCR (14 and 9 cycles, respectively).
Experimental Factor: sampling time point
2015