title: cell2S38
organism: Mus musculus
description: single cell transcriptome sequencing Protocols: 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).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2015-08-02T17:01:21Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-08T23:41:08Z
attributes.tag: External Id
attributes.value: SAMEA3261331
attributes.tag: INSDC center name
attributes.value: EMBL Heidelberg
attributes.tag: INSDC first public
attributes.value: 2015-08-02T17:01:21Z
attributes.tag: INSDC last update
attributes.value: 2018-03-08T23:41:08Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-3346:cell2S38
attributes.tag: batch name
attributes.value: stanford2
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: medullary thymic epithelial cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: facs
attributes.value: PI(neg)CD45(neg)Ly51(neg)EpCAM(pos)MHCII(high)
attributes.tag: individual
attributes.value: cell2S38
attributes.tag: sample name
attributes.value: E-MTAB-3346:cell2S38
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: single cell well quality
attributes.value: OK