Illumina HiSeq 2500 paired end sequencing; Single-cell transcriptome sequencing from mouse mTECs (additional data)
Single-cell transcriptome sequencing from mouse mTECs (additional data)
Human mTECs were processed as in Pinto et al, 2013. Mouse mTECs were isolated and purified as described previously (Rattay et al, 2015). The pre-enriched stromal cell fraction, sorted for total mTECs (n = 259 cells) was stained using the following antibodies: anti-CD45-PerCP (clone 30-F11, BD Pharmingen), anti-EpCAM-Alexa647 (G8.8, described by Farr et al ), anti-CDR1-Pacific Blue or anti-Ly51-FITC (CDR1 hybridoma, described by Rouse et al ; clone 6C3, BD Biosciences).  For the antigen-specific mTECs Tspan8 (n = 48) and Ceacam1 (n = 30) additional antibodies were added to the antibody mix namely: anti-I-A(b)-FITC (clone AF6-120.1, BD Pharmingen), anti-Tspan8-PE (clone 657909, RandD Systems) and anti-CD66a-PE (i.e., anti-Ceacam1, clone CC1, eBioscience). Dead cells were excluded using propidium iodide (PI) in a final concentration of 0.2 ug/ml. Cells were sorted on BD FACSAria(TM) III cell sorter (BD Biosciences) using the single-cell sorting mode as previously described (Derbinski et al, 2008) Human mTEC subsets (surface TRA positive and surface TRA depleted cells) were isolated and FACS sorted as described elsewhere(Pinto et al, 2013). ATAC-seq experiments were performed as reported previously (Buenrostro et al) with the following modifications: 15,000 â 50,000 pooled cells (depending on mTEC subset frequencies) were sorted in PBS (5 percent FCS) and used for ATAC-seq experiments. We used 50 percent of each purified tagmentation reaction for the enrichment PCR (without 5 cycle pre-amplification). Enrichment PCRs were monitored individually using a StepOnePlus Real-Time PCR System (Life Technologies) and the amplification reaction was stopped as soon as amplification approached saturation. After the enrichment PCR, we performed a gel extraction (QIAquick Gel Extraction Kit, QIAGEN) to remove primer dimer.
Experimental Factor: patient3-CEACAM5(pos)
phenotype