MAGE-TAB Version 1.1 Investigation Title Single-cell transcriptome sequencing from mouse mTECs Experimental Design population based design Experimental Design Term Source REF EFO Experimental Design Term Accession Number Experimental Factor Name phenotype individual Experimental Factor Type phenotype individual Experimental Factor Term Source REF Experimental Factor Term Accession Number Person Last Name Reyes Person First Name Alejandro Person Mid Initials Person Email alejandro.reyes@embl.de Person Phone +49 6221 387-8169 Person Fax +49 6221 387-8166 Person Affiliation EMBL Heidelberg Person Address Meyerhofstraße 1, 69117 Heidelberg, Germany Person Roles submitter Person Roles Term Source REF Person Roles Term Accession Number Quality Control Type Quality Control Term Source REF Quality Control Term Accession Number Replicate Type Replicate Term Source REF Replicate Term Accession Number Normalization Type Normalization Term Source REF Normalization Term Accession Number Date of Experiment 2015-02-19 Public Release Date 2015-08-02 PubMed ID 26237553 Publication DOI 10.1038/ni.3246 Publication Author List Brennecke P, Reyes A, Pinto S, Rattay K, Nguyen M, Küchler R, Huber W, Kyewski B, Steinmetz LM Publication Title Single-cell transcriptome analysis reveals coordinated ectopic gene-expression patterns in medullary thymic epithelial cells. Publication Status published Publication Status Term Source REF Publication Status Term Accession Number Experiment Description Promiscuous gene expression (pGE) of tissue-restricted antigens in medullary thyme ephitelial cells (mTECs) is essential for self-tolerance induction and to prevent autoimmunity. We sequenced single mTEC transcriptomes to explore gene expression heterogeneity and to discover patterns of regulation of pGE. Protocol Name P-MTAB-43451 P-MTAB-43452 P-MTAB-43455 P-MTAB-43453 P-MTAB-43454 P-MTAB-43468 Protocol Type sample collection protocol nucleic acid library construction protocol nucleic acid sequencing protocol nucleic acid sequencing protocol nucleic acid library construction protocol normalization data transformation protocol Protocol Term Source REF EFO EFO EFO EFO EFO EFO Protocol Term Accession Number Protocol Description 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). Final multiplexed sequencing libraries were quantified using qPCR and pooled on a MiSeq lane. 76 bp paired-end sequencing was used on a MiSeq Sequencing System (Illumina) and samples yielded between 2,318,942 and 4,184,542 sequenced fragments. We multiplexed 23 samples per Illumina HiSeq 2500 lane and used 100 bp paired-end sequencing. 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 – 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. The ATAC-seq data was mapped to the Human reference genome (ENSEMBL release 75) using GSNAP 2014-07-04. Only uniquely mapping fragments were considered for further analysis. For each annotated transcript in ENSEMBL, we counted of read fragments overlapping with a 4Kb window around the TSS Protocol Parameters Protocol Hardware Illumina MiSeq Illumina HiSeq 2500 Protocol Software Protocol Contact Term Source Name EFO ArrayExpress Term Source File http://www.ebi.ac.uk/efo/ http://www.ebi.ac.uk/arrayexpress/ Term Source Version 2.38 SDRF File E-MTAB-3346.sdrf.txt Comment [Submitted Name] Single-cell transcriptome sequencing from mouse mTECs Comment [SecondaryAccession] ERP009633 Comment [SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/ERR765236-ERR765576 Comment [AEExperimentType] RNA-seq of coding RNA from single cells Comment[ArrayExpressAccession] E-MTAB-3346 Comment [AdditionalFile:fa] ERCC92.fa