MAGE-TAB Version 1.1 Investigation Title Reconstructing the human first trimester fetal-maternal interface using single cell transcriptomics - Smartseq 2 data Experimental Design cell type comparison design Experimental Design Term Source REF EFO Experimental Design Term Accession Number EFO:0001745 Experimental Factor Name single cell identifier Experimental Factor Type single cell identifier Experimental Factor Term Source REF EFO Experimental Factor Term Accession Number Person Last Name Efremova Vento Tormo Henriksson Person First Name Mirjana Roser Johan Person Mid Initials Person Email me5@sanger.ac.uk rv4@sanger.ac.uk mahogny@areta.org Person Phone Person Fax Person Affiliation Wellcome Sanger Institute Wellcome Sanger Institute Wellcome Sanger Institute Person Address Wellcome Genome Campus Hinxton, Cambridgeshire, CB10 1SA. UK Wellcome Genome Campus Hinxton, Cambridgeshire, CB10 1SA. UK Wellcome Genome Campus Hinxton, Cambridgeshire, CB10 1SA. UK 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 2018-03-15 Public Release Date 2019-08-21 PubMed ID 30429548 Publication DOI 10.1038/s41586-018-0698-6 Publication Author List Roser Vento-Tormo, Mirjana Efremova, Rachel A. Botting, Margherita Y. Turco, Miquel Vento-Tormo, Kerstin B. Meyer, Jong-Eun Park, Emily Stephenson, Krzysztof Pola?ski, Angela Goncalves, Lucy Gardner, Staffan Holmqvist, Johan Henriksson, Angela Zou, Andrew M. Sharkey, Ben Millar, Barbara Innes, Laura Wood, Anna Wilbrey-Clark, Rebecca P. Payne, Martin A. Ivarsson, Steve Lisgo, Andrew Filby, David H. Rowitch, Judith N. Bulmer, Gavin J. Wright, Michael J. T. Stubbington, Muzlifah Haniffa, Ashley Moffett & Sarah A. Teichmann Publication Title Single-cell reconstruction of the early maternal-fetal interface in humans Publication Status published Publication Status Term Source REF Publication Status Term Accession Number Experiment Description To explore the interactions between the range of maternal and fetal placental cell types present, we profiled the transcriptomes of more than 50,000 single cells from matched first trimester samples of maternal blood and decidua, as well as fetal cells from the placenta itself. This part contains RNA-seq of Decidua cells using Smartseq 2 Protocol Name P-MTAB-73443 P-MTAB-73439 P-MTAB-73442 P-MTAB-73441 P-MTAB-73440 Protocol Type normalization data transformation protocol sample collection protocol nucleic acid sequencing protocol nucleic acid library construction protocol nucleic acid extraction protocol Protocol Term Source REF EFO EFO EFO EFO EFO Protocol Term Accession Number EFO_0003816 EFO_0005518 EFO_0004170 EFO_0004184 EFO_0002944 Protocol Description Placental and decidual tissue for immunohistochemistry were obtained from elective terminations of normal pregnancies at Addenbrooke’s Hospital between 6 and 12 weeks gestation. Decidual and placental tissue were washed in HAMS F12 medium, macroscopically separated and then washed for at least 10 mins in RPMI or HAMS F12 medium respectively before processing. Decidual tissues were chopped using scalpels into approximately 0.2 mm3 cubes and enzymatically digested in 15ml 0.4mg/mL collagenase V (Sigma, C-9263) solution in RPMI 1640 medium (ThermoFisher Scientific, 21875-034)/10% FCS (Biosfera, FB-1001) at 37°C for 45 min. The supernatant was diluted with medium and passed through 100um cell sieve (Corning, 431752) and then 40um cell sieve (Corning, 431750). The flow-through was centrifuged and resuspended in 5ml of red blood cell lysis buffer (Invitrogen, 00-4300) for 10min. Each first trimester placenta was placed in a petri dish and the placental villi were scraped from the chorionic membrane using a scalpel. The stripped membrane was discarded and the resultant villous tissue was enzymatically digested in 70 ml 0.2% trypsin 250 (Pan Biotech P10-025100P)/0.02% EDTA (Sigma E9884) in PBS with stirring at 37°c for 9 min. The disaggregated cell suspension was passed through sterile muslin gauze (Winware food grade) and washed through with Hams F12 medium (Biosera SM-H0096) containing 20% FBS (Biosera FB-1001). Cells were pelleted from the filtrate by centrifugation and re-suspended in Hams F12. The undigested, gelatinous tissue remnant was retrieved from the gauze and further digested with 10-15 ml collagenase V at 1.0mg/ml (Sigma C9263) in Hams F12 medium/10% FBS with gentle shaking at 37°C for 10 min. The disaggregated cell suspension from collagenase digestion was passed through sterile muslin gauze and the cells pelleted from the filtrate as before. Cells obtained from both enzyme digests were pooled together and passed through 100um cell sieve (Corning, 431752) and washed in Hams F12. The flow-through was centrifuged and resuspended in 5ml of red blood cell lysis buffer (Invitrogen, 00-4300) for 10min. Decidual and blood cells were incubated at 4°C with 2.5ul of antibodies in 1% FBS in DPBS without Calcium and Magnesium (ThermoFisher Scientific, 14190136). DAPI was used for live/dead discrimination. We used an antibody panel designed to enrich for certain population for single-cell sorting and single-cell RNA sequencing (scRNA-seq). Cells were sorted using a Becton Dickinson (BD) FACS Aria Fusion with 5 excitation lasers (355nm, 405nm, 488nm, 561nm and 635nm Red), and 18 fluorescent detectors plus forward and side scatter. The sorter was controlled using BD FACS DIVA software (version 7). For single-cell RNA-seq using the plate-based SS2 protocol, we created overlapping gates that comprehensively and evenly sampled all immune cell population in the decidua. B cells (CD19+ /or CD20+) cells were excluded from our analysis, due to its absence in decidua. Single cells were sorted into 96-well full-skirted Eppendorf plated chilled to 4C, prepared with lysis buffer consisting of 10 ul of TCL buffer (Qiagen) supplemented with 1% b-mercaptoethanol. Single-cell lysates were sealed, vortexed, spin down at 300g at 4C for 1 min, immediately placed on dry ice, and transferred for storage at -80C. The SS2 protocol was performed on single-cells as described previously, with some modifications. Libraries were sequenced aiming at an average depth of 1 million reads/cell, on an Illumina HiSeq 2000 with v4 chemistry (paired-end 75-bp reads). Libraries were sequenced aiming at an average depth of 1 million reads/cell, on an Illumina HiSeq 2000 with v4 chemistry (paired-end 75-bp reads). Libraries were made using Nextera XT RNA was extracted, cDNA created and amplified, as part of the smart-seq 2 protocol. Protocol Parameters Protocol Hardware Illumina HiSeq 2000 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-6678.sdrf.txt Comment [Submitted Name] Reconstructing the human first trimester fetal-maternal interface using single cell transcriptomics - Smartseq 2 data Comment [SecondaryAccession] ERP107748 Comment [SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/ERR3486811-ERR3486811 Comment [SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/ERR2455479-ERR2463075 Comment [AEExperimentType] RNA-seq of coding RNA from single cells Comment[ArrayExpressAccession] E-MTAB-6678