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) We used a modified version of Smrt-seq2. We used 19 cycles of initial PCR amplification and used a ratio of 0.6:1 (instead of a 1:1 ratio) of Ampure XP beads (Beckman Coulter) for the first PCR purification to minimize primer dimer carryover. After the first PCR amplification, cDNA libraries were screened via qPCR (we used a 1:10 dilution of purified cDNA libraries for qPCR reactions) for expression of a mouse housekeeping gene (Ubc), and library size distribution was checked on the Bioanalyzer instrument (Agilent) as reported previously . Only cDNA libraries that passed both quality controls were processed further. We used 100 pg of cDNA for the tagmentation reaction and applied 12 cycles for the final enrichment PCR of adapter-ligated fragments. The last purification step was performed using a 0.8:1 ratio of Ampure SPRIselect beads (Beckman Coulter).
Experimental Factor: PI(neg)CD45(neg)Ly51(neg)EpCAM(pos)MHCII(high)Klk5(qPCR-pos)
phenotype