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Single cell transcriptional characterization of human megakaryocyte lineage commitment and maturation

Single cell RNA-sequencing of sternal bone marrow reciding Hematopoietic Stem Cells (HSCs) and Megakaryocytes (MKs) from individuals undergoing elective open heart valve replacement. HSCs were defined as Lineage-, CD34+, CD38-, CD45RA-, CD90+, CD49f+ cells. MKs where CD41a+, CD42b+ and ploidy was determined with Hoechst. A sternal bone marrow scraping was taken directly following median sternotomy using a Volkmann’s spoon. The sample was collected into an EDTA Vacutainer tube containing 1.8mg/ml EDTA. 4mL of Dulbecco’s phosphate buffered saline (PBS, Sigma) containing 10% human serum albumin (HSA, Gemini Bio Products) was added and the whole volume was resuspended by pipetting 2-3 times. The sample was then put on metallic thermal beads (ThermoFisher Scientific) at a temperature between 0-4°C and transported to the University of Cambridge for further processing. For HSC isolation the cells were stained with the following antibody cocktail: PECy5 conjugated anti-lineage specific antibodies: CD2 (BD), CD3 (BD), CD10 (BD), CD11b (BD), CD11c (BD), CD19 (BD), CD20 (BD), CD56 (BD), biotinylated CD42b (Pab5, NHS Blood and Transplant, International Blood Group Reference Laboratory [IBGRL]), biotinylated GP6 (Pab5, NHS Blood and Transplant, International Blood Group Reference Laboratory [IBGRL]) used in combination with PECy5 conjugated streptavidin (Biolegend). Alexa Fluor 700 conjugated anti-CD34 (BD), PerCP-Cy5.5 conjugated anti-CD38 (BD), Pacific Blue conjugated anti-CD45RA (Invitrogen), PECy7 conjugated anti-CD90 (BD),PE conjugated anti-CD49f (BD). After staining cells were kept at 4°C before sorting using a FACS Aria Fusion flow sorter (BD). Single HSCs defined as Lineage-, CD34+, CD38-, CD45RA-, CD90+, CD49f+ cells were sorted by FACS directly into individual wells of a 96-well plate. Index sort data was collected for each single cell. For MK isolation the cells were stained for surface MK markers with mouse anti-human CD41a APC conjugated antibody (BD) and mouse anti-human CD42b PE conjugated antibody (BD) and for ploidy analysis with 1ug/ml Hoechst 33342 (Invitrogen). After incubation at 37°C for 30 minutes, the cells were kept at 4°C before sorting using a FACS Aria Fusion flow sorter (BD). Single cells and MK pools of 20 cells were sorted by FACS according to ploidy level using a 100uM nozzle directly into individual wells of a 96-well plate. cDNA synthesis and poly(A) enrichment was performed following the G&T-seq protocol (Macaulay et al. 2015), a variation of the Smart-seq2 protocol1. ERCC spike-in RNA (Ambion) was added to the lysis buffer in a dilution of 1:4,000,000.

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The BPS Consortium anticipates that data generated from the project will be used by others, such as required for developing new analytical methods, in understanding epigenetics, for integrative analysis and for other uses. BPS Consortium “Single cell transcriptional characterization of human megakaryocyte lineage commitment and maturation”. Funding for the project was provided by UK Medical Research Council (MR/K024043/1).

THE BAMI PLATELET SUB-STUDY (BPS) CONSORTIUM DATA ACCESS AGREEMENT This agreement governs the terms on which access will be granted through the BAMI Platelet Sub-study Data Access Committee (DAC) to BAMI Platelet Sub-study data available in the European Genome- phenome Archive (EGA) by the European Bioinformatics Institute, the UK Outstation of the European Molecular Biology Laboratory for and mandated by the members of the BAMI Platelet Sub-study Consortium. In signing this agreement, You are agreeing to be bound by the terms and conditions of access set out in this agreement. For the sake of clarity, the terms of access set out in this agreement apply both to the User and the User’s Institution (as defined below). User Institution and User are referred to within the agreement as “You” and “Your” shall be construed accordingly. Definitions: BPS DAC means the BAMI Platelet Sub-study Data Access Committee appointed by the BAMI Platelet Sub-study Consortium to manage access to all data submitted by the BAMI Platelet Sub-study Consortium to the EGA. BAMI Platelet Sub-study Consortium means the consortium of researchers involved in the generation and analysis of the BAMI Platelet Sub-study dataset described in the manuscript “Single cell transcriptional characterization of human megakaryocyte lineage commitment and maturation”. EGA means the European Genome-phenome Archive, a permanent archive of controlled access data generated for biomedical research and deposited to enable data sharing. The EGA is described on the website http://www.ebi.ac.ukl/ega. Data means all and any human genetic or phenotype data obtained from the EGA.
Data Subject means a person, who has been informed of the purpose for which the Data is held and has given his/her informed consent thereto. User means a researcher whose User Institution has previously completed this Data Access Agreement and has received acknowledgement of its acceptance. Publications means, without limitation, articles published in print journals, electronic journals, reviews, books, posters and other written and verbal presentations of research, including social media. User Institution means the organisation at which the User is employed, affiliated or enrolled. Terms and Conditions: In signing this Agreement: 1. You agree to use the Data only for the advancement of medical research and according to the consent obtained from sample donors. 
 2. You agree to preserve, at all times, the confidentiality of information and Data pertaining to Data Subjects. In particular, You undertake not to identify or attempt to identify any Data Subject. 
 3. You agree not to attempt to link the data provided under this agreement to other information or archive data available for the data sets provided, even if access to that data has been formally granted to you, or it is freely available without restriction, without specific permission being sought from the relevant access committees. 4. You agree not to transfer or disclose the Data, in whole or part, or any identifiable material derived from the Data, to others, except as necessary for data/safety monitoring or programme management. Should You wish to share the Data with a collaborator outwith the same Institution, the third party must make a separate application for access to the Data. 
 5. You agree to use the data for the approved purpose and project described in your application; use of the data for a new purpose or project will require a new application and approval. 
 6. You accept that Data will be updated from time to time, with suitable versioning. If the reissue is at the request of sample donors and/or other ethical scrutiny, You will destroy earlier versions of the Data. 
 7. You accept that the BPS DAC, the BPS Consortium, or the funders of the Data or any part of the Data supplied: 
a) bear no legal responsibility for the accuracy or comprehensiveness of the Data; and
b) accept no liability for indirect, consequential, or incidental, damages or losses arising from use of the Data, or from the unavailability of, or break in access to, the Data for whatever reason. 
 8. You understand and acknowledge that the Data may be protected by copyright and other intellectual property rights, and that duplication, except as reasonably required to carry out Your research with the Data, or sale of all or part of the Data on any media may not be permitted. Users of the data are solely responsible for establishing the nature of and complying with any such intellectual property restrictions. 
 9. You recognise that nothing in this agreement shall operate to transfer to the User Institution any intellectual property rights relating to the Data. The User Institution has the right to develop intellectual property based on comparisons with their own data. 
 10. You accept that this agreement will terminate immediately upon any breach of this agreement by You and You will be required to destroy any Data held. 
 11. You accept that it may be necessary for the BPS Consortium or its appointed agent to alter the terms of this agreement from time to time in order to address new concerns. In this event, the EGA or its appointed agent will contact You to inform You of any changes and You agree that Your continued use of the Data shall be dependent on the parties entering into a new version of the Agreement. 
 12. You agree that you will submit a report to the BPS DAC, if requested, on completion of the agreed purpose. The BPS DAC agrees to treat the report and all information, data, results, and conclusions contained within such report as confidential information belonging to the User Institution. 
 13. You accept that the Data may be protected by and subject to international laws, including but not limited to applicable laws on the protection of personal data, and that You are responsible for ensuring compliance with any such applicable law. You undertake to implement or have implemented adequate technical and organisational security measures to protect Data. The BPS DAC reserves the right to request and inspect data security and management documentation to ensure the adequacy of data protection measures, particularly in countries that have no national laws comparable to those which pertain in the EEA. 
 BPS DAA ver1, un 2019 APPENDIX 1 BPS Publications Policy The BPS Consortium anticipates that data generated from the project will be used by others, such as required for developing new analytical methods, in understanding epigenetics, for integrative analysis and for other uses. Authors who use data from the project must acknowledge the BPS Consortium using the following wording "This study makes use of data generated by the BPS Consortium. A full list of the investigators who contributed to the generation of the data is available in “Single cell transcriptional characterization of human megakaryocyte lineage commitment and maturation”. Funding for the project was provided by UK Medical Research Council (MR/K024043/1). Users must also cite the primary BPS publication mentioned above. Users should note that the BPS Consortium bears no responsibility for the further analysis or interpretation of these data, over and above that published by the Consortium. BPS DAA ver1, un 2019

Studies are experimental investigations of a particular phenomenon, e.g., case-control studies on a particular trait or cancer research projects reporting matching cancer normal genomes from patients.

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EGAS00001004844 Other
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